Download Patterns of Infection:a Delicate Balance

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Introduction
Infection Strategies
Life Cycles and Patterns of Viral
Infections
Initiating an Infection
Basic Requirements
Tropism
Successful Infections Must Evade Host
Defenses
Many Other Variables Govern the Result
of Infection
Acute Infections
Definition and Requirements
Acute Infections Present Common Public
Health Problems
Defense Against Acute Infections
Multiple Acute Infections in a
Single Host
Pathogenic Effects of an Acute Infection
Persistent Infections
Definition and Requirements
Infections of Tissues with Reduced Immune Surveillance
Direct Infection of the Immune System
Itself
Two Viruses That Cause Persistent
Infections
Latent Infections
An Extreme Variation of the Persistent
Infection
Two Viruses That Produce Latent
Infections
Slow Infections
Sigurdsson’s Legacy: Icelandic Sheep and
Fatal Degenerative Diseases
Slow Viruses and “Unconventional
Agents”
Other Patterns of Viral Infections
Abortive Infections
Transforming Infections
Perspectives
References
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Patterns of Infection: a Delicate Balance
You know something’s happening, but you
don’t know what it is, do you, Mr. Jones?
R. ZIMMERMAN
Introduction
Infection Strategies
Viral infections of individuals in populations differ from viral infections of tissue culture cells in the laboratory. In the former, initiation of the infection, and
its eventual outcome, rests upon complex variables such as host defenses and
the environment. Despite such complexity and the plethora of viruses and
hosts, common patterns of infection appear. In general, natural infections can
be rapid and self-limiting (acute infections) or long-term (persistent infections). Variations and combinations of these two modes abound. While we
can provide detailed descriptions of individual patterns of infection, we are in
the early days of understanding the molecular mechanisms required to initiate
or maintain any specific one.
Life Cycles and Patterns of Viral Infections
A cursory examination of the animal viruses that grow in cultured cells identifies many distinctive life cycles with common features. Some viruses rapidly
kill the cell while producing a burst of new infectious particles (cytopathic
viruses). Others infect cells and actively produce infectious particles without
causing immediate host cell death (noncytopathic viruses). Alternatively,
some viruses infect but neither kill the cell nor produce any virus progeny.
These apparently diverse life cycles defined in vitro comprise the two primary
patterns of infection in the host: acute and persistent infections (Fig. 15.1).
Variations on these archetypes occur repeatedly. For example, a latent infection is an extreme version of a persistent infection. Similarly, slow, abortive,
and transforming infections are more complicated variants of a persistent
infection.
Before we discuss patterns of infection, we will review the parameters important in initiating and establishing any viral infection.
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Acute infection
• Rhinovirus
• Rotavirus
• Influenza virus
Virus production
Persistent infection
• Lymphocytic
choriomeningitis virus
Death
Latent, reactivating infection
• Herpes simplex virus
Slow virus infection
• Measles SSPE
• Human immunodeficiency
virus
Time
Death
Figure 15.1 General patterns of infection. Relative virus production is plotted as a function of time
after infection. The time when symptoms appear is indicated by the red shaded area, and the period
in which infectious virus is released (available to infect other hosts) is indicated by the bracket. The
top panel is the typical profile of an acute infection, in which virus is produced, symptoms appear, and
virus is cleared within 7 to 10 days after infection. The second panel is the typical profile of a persistent infection, in which virus production continues for the life of the host. Symptoms may or may not
appear just before death, depending on the virus. Infectious virus is usually produced throughout the
infection. The bottom two panels are variations of the persistent infection. The third panel depicts a
latent infection, in which an initial acute infection is followed by a quiescent phase and repeated bouts
of reactivation. Reactivation may or may not be accompanied by symptoms but generally results in the
production of infectious virus. The fourth panel depicts a slow virus infection, in which a period of
years intervenes between a typical primary acute infection and the usually fatal appearance of symptoms. Depending on the virus, the production of infectious virus during the long period between primary infection and fatal outcome may be continuous (e.g., human immunodeficiency virus) or absent
(e.g., measles virus subacute sclerosing panencephalitis [SSPE]). The brackets indicating infectious
virus release are placed arbitrarily to indicate this phenomenon. Adapted from F. J. Fenner et al., Veterinary Virology (Academic Press, Inc., Orlando, Fla., 1993), with permission.
Initiating an Infection
Basic Requirements
Three requirements must be met to ensure successful infection in an individual host: sufficient virus must be available to initiate infection, the cells at the site of infection
must be susceptible and permissive for the virus, and the
local host antiviral defense systems must be absent or at
least initially ineffective.
The first requirement erects a substantial barrier to any
infection and forms a significant weak link in the transmission of infection from host to host. Free virus particles
face both a harsh environment and rapid dilution that can
reduce their concentration. Viruses spread by contami-
nated water and sewage must be stable in the presence of
osmotic shock, pH changes, and sunlight and must not adsorb irreversibly to debris. Aerosol-dispersed viruses must
stay wet and highly concentrated to infect the next host.
Such viruses do best in populations in which individuals
are in close contact. Viruses that are spread by biting insects, contact of mucosal surfaces, or other means of direct
contact, including contaminated needles, have little environmental exposure.
Even if one virus particle survives the passage from one
host to another, infection may fail simply because the concentration is not sufficient. In principle, a single virus particle should be able to initiate an infection, but host
physical and immune defenses, coupled with the com-
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Patterns of Infection: a Delicate Balance
plexity of the infection process itself, demand the participation of many particles. How many particles are required
to initiate and maintain an infection? Unfortunately, there
is no simple answer to this question because the success of
an infection depends on the particular virus, the site of infection, and the physiology and age of the host. However,
some basic facts help to guide us.
Statistical analysis of infections in cultured cells demonstrates that on average a single virus particle can initiate an
infection, but that many perfectly competent virions fail to
complete this process. Such failure can be explained in
part by the complexity of the infectious cycle: there are
many distinct reactions, and the probability of a virus particle completing any one is not 100%. For example, virus
particles face many potentially nonproductive interactions
with debris and extracellular material during their initial
encounter with the cell surface. Even if a virus attaches
successfully to a permissive cell, it may be delivered to a
digestive lysosome upon entry. It is not that any of the
steps in infection are inherently inefficient, but rather that
many of these false starts or inappropriate interactions are
irreversible, aborting infection by the virion.
In addition, populations of viruses often contain particles
that are not capable of completing an infectious cycle. For
example, defective particles can arise from mistakes during
virus replication or from interaction with inhibitory compounds in the environment. In the laboratory, a quantitative measure of the proportion of infectious viruses is the
particle to plaque-forming unit (PFU) ratio. As described in
chapter 2, the number of physical particles in a given preparation is counted, usually with an electron microscope, and
compared to the number of infectious units or PFU per unit
volume. This ratio is a useful indicator of the quality of a
virus preparation, as it should be constant for a given virus
prepared by identical or comparable procedures.
Tropism
All patterns of infection are dominated by the property of
viral tropism. Tropism is a predilection of viruses to infect
certain tissues and not others. For example, an enterotropic virus replicates in the gut, whereas a neurotropic virus replicates in cells of the nervous system.
Some viruses are pantropic, infecting and replicating in
many cell types and tissues. Tropism may be determined
by the distribution of receptors for entry (susceptibility),
it may be the result of a requirement of the virus for differentially expressed intracellular gene products to complete the infection (permissivity), or it may indicate that
the virus is physically prevented from interacting with tissues that otherwise could support virus growth. In most
cases, tropism is determined by a combination of two or
more of these parameters.
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Tropism affects the pattern of infection, pathogenesis,
and long-term virus survival. The role of tropism in pathogenesis will be discussed in greater detail in chapter 17.
Consider the example of herpes simplex virus, an alphaherpesvirus. This human herpesvirus is often said to be
neurotropic because of its noteworthy ability to infect and
reactivate from the nervous system. But in fact, herpes
simplex virus is pantropic and replicates in many cells and
tissues in the host. By infecting neurons, it establishes a
stable latent infection (see “Two Viruses That Produce
Latent Infections” below), but because it is pantropic it
spreads to other tissues and host cells. One serious consequence is that if the virus escapes host defenses at the site
of infection, it will spread widely, causing disseminated
disease, as can occur when herpes simplex virus infects babies and immunocompromised adults. Herpes simplex
virus neurotropism leads to yet another serious result of
infection. On rare occasions, this virus can enter the central nervous system and cause an encephalitis which is
often fatal.
Similar alterations of tropism in a single virus infection
can be observed for many RNA viruses. Consider poliovirus, a picornavirus with enteric tropism. The poliovirus
receptor is found on the surface of almost every cell of the
body, but in the common natural infection, poliovirus
replicates in the gut; it rarely spreads to other tissues. It is
efficiently spread from host to host by fecal-oral transfer.
As an enteric virus, poliovirus is not usually a major health
problem. However, neurotropic strains of poliovirus that
spread from the gut to motor centers of the central nervous system arise. Infections by such viruses result in
paralysis and often death, a considerably more serious aspect of poliovirus infection. At least two questions arise.
What are the molecular changes in the virus that alter its
tropism and virulence? Is there a selective advantage of
the change in tropism for virus survival? We have some
data with regard to the former question (see chapter 17
and Fig. 19.6B) but can only speculate about the latter.
Successful Infections Must Evade
Host Defenses
The sites of virus entry in a host are described in chapter
17. To initiate an infection at these sites, viruses must
counter the host defenses by an active or passive mechanism or by a combination of both. This is a simple but
often overlooked fact; most of the information encoded in
viral genomes never has a chance to be expressed because
most infections are blocked before anything happens.
Moreover, the physical defenses exemplified by skin and
mucous surfaces, in combination with the innate defenses
described in chapters 14 and 17, may block or limit infection before the acquired immune system is activated.
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Some of these defenses may be overcome passively by
an overwhelming inoculum of virus falling on a mucosal
surface. Single droplets found in the aerosol produced by
sneezing can contain as many as 100 million rhinovirus
particles; a similarly high number of hepatitis B virus particles can be found in 1 ml of blood from a hepatitis patient.
At these concentrations, it may be impossible for physical
and innate defenses to block every infecting virus particle.
Free passage of virus through the primary physical barriers of skin and mucous layers made possible by a cut, abrasion, or needle stick may also allow passive evasion of
defenses. A more egregious breech of both primary and
secondary defenses may occur during organ transplantation, which places viruses in direct contact with potentially
susceptible cells in immunosuppressed patients. Some
viruses (e.g., herpesviruses, papovaviruses, and rabies
virus) adopt a passive stance simply by infecting organs or
cells not exposed to antibodies or cytotoxic lymphocytes.
Many viruses have evolved active mechanisms for bypassing or disarming host defenses (Table 15.1). For example, some viruses express proteins that block the general
suicide program activated in most virus-infected cells
(apoptosis), while the high mutation rates of most RNA
viruses result in the production of altered proteins that
allow the virus to evade immune defenses.
Apoptosis (Programmed Cell Death)
It is axiomatic that viruses require an intact and functioning cell during the initiation of infection because premature demise of the cell would effectively block virus
Table 15.1 Active evasion of immune defense
Process
Exchange of genetic information
by reassortment of genomic
segments to replace entire
surface proteins
Blocking of specific immune
defenses
Initiation of a noncytopathic
infection to delay or avoid a
robust immune response
Virus examples
Influenza virus, rotavirus
Adenovirus, herpesviruses,
poxviruses, human immunodeficiency virus
Arenaviruses, paramyxoviruses, polyomavirus,
papillomaviruses, parvoviruses
replication and spread. Therefore, it should not be surprising that cell death is a frequent antiviral defense. When
the biochemical alterations initiated by viral infection are
detected, a process of self-destruction called apoptosis is
initiated (Fig. 15.2). This pathway was first discovered in
1842 by Carl Vogt, and the term “apoptosis” was coined in
1972. This process is controlled by a variety of interacting
signals that monitor the orderly processes of growth regulation, cell cycle progression, and metabolism in metazoan
cells. Survival signals from the cell’s environment and internal signals reporting on cell integrity normally keep the
apoptosis response in check. When these signals are perturbed, cell death invariably ensues (Fig. 15.3). Apoptosis
can be activated by a large variety of both external and internal stimuli. As an example of the former, cytotoxic
T cells may kill their target by apoptosis initiated by Fas
Figure 15.2 Apoptosis, the process of programmed cell death. Apoptosis can be recognized by several distinct changes in cell structure. A normal cell is shown at the left. When
programmed cell death is initiated, as indicated by the second cell, the first event visible is
the compaction and segregation of chromatin into sharply delineated masses that accumulate at the nuclear envelope (dark blue shading around periphery of nucleus). The cytoplasm also condenses, and the outline of the cell and nuclear membranes changes, often
dramatically. The process can be rapid, so that within minutes the nucleus fragments and
the cell surface convolutes, giving rise to the characteristic “blebs” and stalked protuberances illustrated. These blebs then separate from the dying cell and are called apoptotic bodies. Macrophages (the cell at the right) engulf and destroy these apoptotic bodies. Adapted
from J. A. Levy, HIV and the Pathogenesis of AIDS, 2nd ed. (ASM Press, Washington, D.C.,
1998), with permission.
Apoptotic
bodies
Normal cell
Apoptosis begins
Macrophage
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Patterns of Infection: a Delicate Balance
Ad E1A
HPV E7
SV40 Tag
Transcription and cell
cycle control
Developmental signals
p53
Ad E4ORF6
Ad E1B-55K
HPV E6
SV40 Tag
HBV pX
EBV BZLF1
HIV Tat
HTLV-1 Tax
Fas ligand increased
Ad E3 14.7K
Ad E3 10.4/14.5K
Fas
Growth
arrest
EBV LMP-1
Bcl-2 family members
control apoptosis
Ad E1B 19K
EBV BHRF1
ASFV 5-HL
KSbcl2
Cytokines:
Tnf-α/Tnfr
DNA damage
Activate
apoptosis
Death domain protein
signalling complexes
Extracellular signals
??
Ad E1A/E4
Ad E3 11.6K
Ad E4
HPV E7
CAV apoptin
Viral inhibitors
of apoptosis
Myxoma virus T2
Bax
Caspases
Cellular
inhibitors of
apoptosis
523
Il-1β
Lamins and
other substrates
Cowpox virus CrmA
Baculovirus p35
Vaccinia virus SPI-2
Inflammation
Apoptosis
Figure 15.3 Regulation of apoptosis by cellular and viral gene products. Flow chart of the major
processes and selected signals in the pathway mediating cell growth and programmed death (tan). A
variety of signals from the cell cycle, developmental pathways, and DNA damage activate p53 (dark
red boxes). Activated p53 participates either in growth arrest or apoptosis (blue). Apoptosis can be abrogated by a variety of cellular regulatory proteins illustrated here by Bcl-2 family members. Expression of these gene products is affected by a variety of extracellular signals (purple box) such as
cytokines like tumor necrosis factor (Tnf) and the interaction of cell surface ligands and receptors, including Fas ligand and Fas receptor. If Bcl-2-like proteins do not block the apoptotic pathway, then cysteine proteases (caspases) are activated and act in casade to carry out the final stages of apoptosis. One
such caspase family member is caspase 1, the interleukin-1b-converting enzyme (ICE) protease. This
enzyme activates interleukin-1b, which in turn facilitates inflammation (blue). Other caspases cleave
a variety of substrates, including lamins and pronucleases. As a result of caspase action, the cell architecture is altered and cellular DNA is degraded. Viral proteins known to affect the pathway of apoptosis are indicated in the white boxes. Green arrows indicate induction, stimulation, or activation; red
bars indicate inhibition. Abbreviations: HTLV-1, human T-cell leukemia virus type 1; SIV, simian immunodeficiency virus; HIV, human immunodeficiency virus; CAV, chicken anemia virus; HPV, human
papillomavirus; Ad, adenovirus; SV40, simian virus 40; ASFV, African swine fever virus; HBV, hepatitis B virus; EBV, Epstein-Barr virus; KS, Kaposi’s sarcoma herpesvirus (human herpesvirus 8); Tnfr, Tnf
receptor; Tag, large T antigen.
ligand on the T cell binding to Fas receptor on the target
cell. Similarly, apoptosis is initiated when the cytokine
tumor necrosis factor (Tnf) binds to its receptor on a virusinfected cell. Common intracellular initiators include DNA
damage and ribonucleotide depletion. In these situations,
the cell cycle regulatory protein p53 is activated (see chapter 16) and apoptosis ensues. How activated p53 stimulates this process is still unresolved. Regardless of the
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nature of the initiation signal, the result is activation of
common effectors, the caspases. Caspases are members of
a family of cysteine proteases that specifically cleave proteins after asparagine residues. They carry out limited proteolysis of many cellular substrates in a protease cascade,
not unlike blood clotting or the complement cascade. The
principle is similar: a modest initial signal can be amplified
significantly, culminating in an all-or-none result. In apoptosis, after caspases are activated (no matter how diverse
the initial activating signal), the end results are always the
same: cell and organelle dismantling, vesicle and membrane bleb formation, and DNA cleavage to nucleosomesized fragments (Fig. 15.4). As one might expect, apoptosis
is a tightly regulated process, and the cell encodes several
suppressors including members of the Bcl-2 family. Despite the complexity, it is best to think of apoptosis as a default pathway held in check by the continuous action of a
variety of regulatory molecules.
While apoptosis can be a powerful antiviral defense, it
is also a normal host process essential for orderly development of many organisms. Indeed, cell-encoded inducers of
apoptosis (e.g., Bax and Bad [Fig. 15.3; Table 15.2]) must
be synthesized for the maintenance of important aspects of
normal cell physiology and homeostasis, such as the regulation of cell numbers in development and proper functioning of the immune system. Apoptosis also has an
important role in presenting antigens to cytotoxic T cells.
When influenza virus-infected cells undergo apoptosis, the
cellular debris containing viral antigens is ingested by dendritic cells which present the antigen to T cells. If apoptosis is blocked in the infected cells, dendritic cells do not
pick up antigen and T cells are not activated.
Like all cellular antiviral defenses, apoptosis can be a
double-edged sword. Viruses have evolved to survive despite apoptosis (Box 15.1). Some viruses are able to bypass
it by synthesizing proteins that interfere with the program
at a number of distinct steps (see below; Fig. 15.3). Others
actually incorporate apoptosis as part of their life cycles.
Most DNA viruses induce apoptosis upon infection because they require all or part of the host’s transcription,
translation, and replication machinery (Fig. 15.3). In
many infections, the target cell is quiescent and hence unable to provide the enzymes and other proteins needed by
the infecting virus. Consequently, to replicate in these
cells, the virus must actively engage the host’s cell cycle
machinery and induce the cell to leave the resting state.
However, cell cycle checkpoint proteins then respond to
these unscheduled events by inducing apoptosis (Fig.
15.3). Typically, activation of the cell cycle is accomplished
by viral early proteins (e.g., adenovirus E1A proteins or
simian virus 40 [SV40] large T antigen). The genomes of
many viruses that activate apoptosis as part of their life
cycle carry additional genes whose products block this potentially lethal process long enough for the virus to replicate its genome and produce infectious particles.
Viral proteins that inhibit apoptosis. Viral mutants
unable to inhibit apoptosis were detected originally because the host DNA of mutant infected cells was unstable,
the cells lysed prematurely, and, as a consequence, viral
yields were reduced, resulting in small plaques. The mutant gene products are analogs of host Bcl-2, caspase inhibitors, and other proteins that prevent or delay apoptotic
death of infected cells (Fig. 15.3; Table 15.1).
Some viral proteins that inhibit apoptosis have rather
remarkable properties. For example, the adenovirus E1B
19K protein inhibits apoptosis by at least two distinct
mechanisms. In one, the protein binds to the apoptosispromoting Bax protein to prevent caspase activation. In
the other, the protein interferes with the function of adaptor molecules that interact with and activate caspases.
Some herpes- and poxviruses encode other types of apoptosis inhibitors. Death receptors are cell surface proteins
that transmit apoptosis signals on binding death ligands.
These receptors are part of the tumor necrosis factor receptor gene superfamily. A characteristic of such receptors
is a short amino acid sequence, called the death domain,
which defines a surface of the cytoplasmic portion of the
receptor that engages the cell’s apoptotic machinery when
the appropriate ligand is bound. Cellular adaptor proteins
that bind this domain in this way include Fas-associated
death domain protein. This complex of Fas-associated
death domain protein and tumor necrosis factor or Fas receptor then activates caspase 8 to initiate the caspase cascade. Computer searches of viral DNA sequences revealed
several viral proteins with sequences similar to the death
domain of cellular death receptors. The viruses expressing
such proteins are found in two DNA virus groups, the
gammaherpesviruses (e.g., human herpesvirus 8, equine
herpesvirus 2, and bovine herpesvirus 4) and the
poxviruses (e.g., molluscum contagiosum virus). When
these viral proteins were expressed in cells, they blocked
Fas ligand- and Tnf-induced apoptosis. Work is now in
progress to determine the role of these novel apoptosis inhibitors in natural infections.
RNA virus infections are also modulated by apoptosis.
In the case of Sindbis virus, apoptosis directs the pattern of
infection. In some vertebrate tissue culture cell lines, Sindbis virus infection is acute and cytopathic because apoptosis is induced. However, the virus establishes a persistent
infection of postmitotic neurons in culture because the cell
death pathway is not activated. A corollary of the in vitro
experiment can be observed in animals. When virus is injected into an adult mouse brain, it establishes a persistent
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Figure 15.4 Apoptosis in a CD41 T cell from peripheral
blood. (A) Agarose gel electrophoresis of total T-cell DNA
after treatment of cells with various antibodies (lanes 2, 3,
and 5) or gamma irradiation (lane 4). Lane 1 contains DNA
from untreated cells. The typical ladder pattern of DNA digestion associated with apoptosis is observed after gamma irradiation. (B) Ultrastructural morphology of a normal CD41 T cell
(a) and a cell in early stages of apoptosis (b) and terminal
stages of apoptosis (c). Bar 5 1 mm. Reprinted from J. A. Levy,
HIV and the Pathogenesis of AIDS, 2nd ed. (ASM Press, Washington, D.C., 1998), with permission.
noncytopathic infection, but in neonatal mouse brains, the
virus is cytopathic and lethal. Sindbis virus induces lethal
apoptosis in neonatal mouse brains because cellular inhibitors of apoptosis are not produced in developing neurons as they are in adult neurons.
Antiviral Cytokine Defense
Viral infection invariably stimulates cells to make interferons and cytokines. These molecules are parts of the host
primary innate defense system. To establish an infection,
some viruses, particularly cytopathic viruses, produce cytokine antagonists to counteract this most potent early host
defense. For example, poxvirus genomes encode a variety
of anti-immune defense proteins including soluble receptors that bind many host cytokines so that the cytokines
cannot reach their proper receptors on cells (Fig. 15.5; see
also Table 14.1). Remarkably, soluble gamma interferon
(Ifn-g) receptors are produced by at least 17 orthopoxviruses; obviously, this cytokine has an important role in
thwarting poxvirus infection. These decoy cytokine receptors are examples of the so-called unselfish defense,
as their presence affects the entire population of infecting
viruses and microbes, not just the virus that directs their
synthesis.
Tumor necrosis factor is a multifunctional cytokine produced primarily by activated monocytes and macrophages.
It can induce an antiviral response when it binds to receptors on virus-infected cells. Such cells are especially sensitive to tumor necrosis factor, most likely because viral
infection blocks or alters host protein synthesis. Within
seconds, the combination of infection and binding of
tumor necrosis factor to its receptor initiates a signal transduction cascade that activates caspases and thus apoptosis.
Many adenoviruses can counteract the lytic effect of
tumor necrosis factor with several small proteins encoded
in the E3 region of the genome (Fig. 15.6; Table 15.3).
Antigenic Variation
Antigenic variation, which results when viruses interact
with the immune system, is an important mechanism of
virus evolution and survival (Box 15.2). Invariably, muta-
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Table 15.2 Selected viral and cellular regulators of apoptosis
Gene product(s)
Inducers
Viral
Viral infection (general)
Adenovirus E1A, E4, E3, SV40 large T,
human papillomavirus E7
HIV, simian immunodeficiency virus Tat
Parvovirus B19 NSP
Chicken anemia virus VP3
Cellular
c-myc protein
Tumor necrosis factor
Fas antigen
p53 protein
Inhibitors
Cellular
Bcl-2
BclXL
Viral
Adenovirus E1B 19K
Adenovirus E1B 55K
Adenovirus E3 14.7K
Epstein-Barr virus latent membrane
protein 1
Ksbcl-2
Herpesvirus saimiri ORF16
Human cytomegalovirus IE1/IE2
Baculovirus p35
Baculovirus IAP protein
Poxvirus CrmA
Hepatitis B virus pX
Function
DNA damage, host shutoff
Cell cycle control, proliferation, transformation, transcription
Transcription
Nonstructural protein, replication, transcription
Structural protein
Proliferation, transformation, transcription
Antiviral and antitumor activities
Negative selection of lymphocytes
Cell cycle control, tumor suppressor, transcription
Blocks apoptosis in hematopoietic cells in response to growth factor withdrawal, CD3,
irradiation, glucocorticoids; maintains B-cell memory; role in T-cell maturation
Like Bcl-2, but functions in neurons
Blocks apoptosis by tumor necrosis factor, Fas, p53; transformation
Inactivates p53; transformation
Blocks apoptosis by tumor necrosis factor
Increases expression of Bcl-2; latency, transformation
Human herpesvirus 8 Bcl-2 homolog
Bcl-2 homolog
Immediate-early proteins 1 and 2; block apoptosis induced by tumor necrosis factor alpha
and adenovirus E1A but not that induced by UV irradiation
Inhibits apoptosis in vertebrate and insect cells; inhibits interleukin-1b-converting enzyme
(ICE-like family) protease
Inhibits apoptosis by a different mechanism than p35
Serpin; serine protease inhibitor; ICE protease inhibitor
Blocks p53-mediated apoptosis
tions accumulate as viruses replicate, and, depending on
the selective forces to which they are exposed, some mutants propagate while others are eliminated. In an immunocompetent host, viral antigenic variation comes
B OX 1 5 . 1
The many ways in which
viruses perturb apoptotic
pathways
about by two distinct processes called antigenic drift and
antigenic shift. Antigenic drift is the appearance of virus
with a slightly altered surface protein (antigen) structure
following passage in the natural host. Mutants expressing
At the cell surface
Apoptosis-inducing cytokines/receptors (e.g., receptor mimicry)
Membrane disruption (e.g., virion adsorption/engagement of receptors)
In the cytoplasm
Metabolic inhibitor (e.g., arrest of
host translation)
Cytoskeletal modification (e.g., disruption of actin microfilaments)
Signal transduction blocker (e.g.,
death domain proteins)
In the nucleus
Host DNA degradation
Altered gene expression (e.g., increased expression of heat shock
genes)
Miller, L. K., and E. White (ed.). 1998. Apoptosis in viral infections. Semin. Virol. 8:443–523.
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Patterns of Infection: a Delicate Balance
Poxvirus-infected cell
Block intracellular
action of Ifn
Block ICE (pro-Il-1β
converting enzyme)
C3b and C4b
complement
binding proteins
Ifn-α and -β soluble
receptor
Ifn-γ soluble
receptor
Il-1β soluble
receptor
Replicating virus
Tnf-α soluble
receptor
3-β-Hydroxysteroid
dehydroxygenase
Block MHC
class I peptide
presentation
Steroid hormone
Nucleus
Cytoplasm
Figure 15.5 Immune evasion mechanisms encoded by
poxviruses. After infection of a cell by a poxvirus, a variety
of viral proteins that influence host defenses are produced.
Several examples of proteins that block different arms of the
host response to infection (e.g., complement activation, cytokine function, antigen presentation to cytotoxic T lymphocytes, inflammation) or mimic host cytokines or cytokine
receptors are represented. Modified from A. Alcami and G. L.
Smith, Immunol. Today 16:474–478, 1995, with permission.
527
altered surface proteins often escape the host immune response and are propagated in the population. In contrast,
antigenic shift is a major change in the surface protein of
a virus as completely new surface proteins are acquired by
the virus. This process occurs when viruses with segmented genomes exchange segments after coinfection.
The new reassortant viruses display dramatic changes in
surface proteins that facilitate escape from immune surveillance (Box 15.3).
As surface proteins are often absolutely essential for the
attachment and entry of viruses into cells, their function
cannot be compromised during antigenic variation. Some
viruses, like human immunodeficiency virus and influenza virus, are more tolerant of antigenic variation than
others and are said to be plastic or to have structural
plasticity. In contrast, nonenveloped RNA viruses, like
poliovirus, can tolerate only modest antigenic drift in their
capsid proteins. Capsid proteins, unlike envelope proteins,
participate in multiple protein-protein and protein-nucleic
acid interactions required to package the genome and to
assemble or disassemble the icosahedral capsid. Mutations
that reduce antibody binding during antigenic drift are
likely to affect these processes and thus are often detrimental to virus reproduction.
Antigenic drift in human immunodeficiency virus infections. Virus-infected individuals produce virus for
years before they develop the symptoms of AIDS. In the
early stages of infection, more than 109 new virus particles
are produced in an infected patient each day, and these
viruses are confronted continually by the immune system.
However, the virus is not completely eliminated, as witnessed by the continuing replication of virus for years.
Until the final stages of AIDS, the immune system continually selects viruses that can grow from the pool of newly
replicated virus. At these rates of replication and selection,
progeny viruses are many generations removed from the
Table 15.3 Functions of adenovirus E3 proteinsa
Host
response
CTL
gp19K
ER membrane
Tnf
14.7K
Nucleus and cytoplasm
Plasma membrane
Egf/Fas
RIDa (10.4K),
RIDb (14.5K)
RIDa (10.4K),
RIDb (14.5K)
ADP (11.6K)
Cell lysis
a
Protein
Intracellular location(s)
Plasma membrane
Nuclear membrane
Function or mechanism
Binds to MHC class I antigens; blocks their transport from ER to cell
surface; prevents CTL recognition
Prevents Tnf cytolysis; inhibits Tnf-induced inflammatory response
by blocking activation of phospholipase A2
Prevents Tnf cytolysis by a different mechanism than 14.7K
Stimulates internalization and degradation of Egf receptor; inhibits
Fas agonist-induced apoptosis
Promotes release of virus from lysed cells
See also Fig. 15.6. Abbreviations: CTL, cytotoxic T lymphocyte; MHC, major histocompatibility complex; ER, endoplasmic reticulum; Tnf, tumor necrosis factor;
Egf, epidermal growth factor.
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Adenovirus
Death
Protein
12.5K
6.7K
Function
unknown
gp19K
Inhibits
killing by
cytotoxic
T cells
ADP
Receptor
Internalization and
Degradation
RID complex
RIDα
Promotes
virus
release
RIDβ
Inhibits:
• Tnf-induced
apoptosis
14.7K
Inhibits:
• Tnf-induced
apoptosis
• Tnf-induced
• Inflammation
translocation of
cPLA2 to membranes
• Fas agonistinduced apoptosis
• Inflammation
Reduces expression of
Fas and epidermal
growth factor receptor
Figure 15.6 The adenovirus type 2 E3 region, a cluster of seven genes encoding proteins that
mediate host defense evasion. The proteins were named initially according to their apparent molecular masses (e.g., 14.7K for 14.7 kDa). Recently, some of the proteins have been given names that reflect their known functions. RID is an acronym for “receptor internalization and degradation.” The RID
protein complex (previously called E3-10.4K/14.5K) is composed of RIDa (10.4 kDa) and RIDb (14.5
kDa). RID proteins have multiple functions, as indicated in the figure. These functions may or may not
represent the same molecular mechanism. ADP is an abbreviation for “adenovirus death protein.”
Even though the ADP gene is in the E3 cluster, it is expressed as a late gene by alternative splicing from
the major late promoter. ADP promotes cell lysis and virus release after virus replication is complete.
Integral membrane proteins are indicated by reddish shading. gp19K is a glycoprotein that reduces
MHC class I protein expression and inhibits killing by cytotoxic T cells. The 14.7K protein inhibits Tnfinduced apoptosis. The functions of the 12.5K and 6.7K proteins have not been identified. Adapted
from W. S. M. Wold and A. E. Tollefson, Semin. Virol. 8:515–523, 1998, with permission.
original infecting virus and soon comprise a variety of mutants selected in part through the process of antigenic drift.
We will discuss the pathogenesis of human immunodeficiency virus more fully in chapter 18.
Such antigenic drift also is believed to facilitate escape
from immune surveillance by a second mechanism, a response to the high variation of individual viral peptides
presented on the surfaces of infected cells by major histocompatibility complex (MHC) class I molecules. Some cytotoxic T cells specific for a given viral peptide recognize
the cognate mutant peptide on infected cells, but because
the peptides are similar but not identical, the cytotoxic T
cell is inactivated, or “anergized.” Instead of being destroyed, the infected cell actually inactivates a cytotoxic T
cell that could have destroyed other cells containing different virus mutants. This mechanism of inactivating T
cells by antigen “mimics” or “decoys” is likely to function
for any virus exhibiting high rates of antigenic drift.
Interference with Expression and
Function of MHC Proteins
The cytotoxic-T-cell response is one of the most powerful adaptive host defenses against viral infection. This response depends in part on the ability of host T cells to
detect viral antigens on the surfaces of infected cells and to
kill these cells. The recognition of infected cells requires the
presentation of viral peptides by MHC class I proteins. The
pathway by which peptide antigens are produced and presented on the infected cell surface is discussed in chapter 14
(Fig. 14.14). Obviously, any viral strategy that stops viral
peptides from appearing on MHC class I molecules on
the surface of cells provides an important selective advantage.
Peptide presentation by MHC class I can be reduced by
lowering expression of the MHC genes directly, by interfering with the production of peptides by the proteasome,
or by interfering with subsequent assembly and transport
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B OX 1 5 . 2
Influenza, which has been recognized
as a human disease for centuries, is notable
because although it causes a typical acute
infection and elicits a strong immune response, it continues to occur regularly in
the population. Influenza can occur in
many nations almost simultaneously (pandemics) with serious consequences.
After more than 65 years of work, we
now have some reasonable ideas why this
virus causes epidemics of “flu” annually
and pandemics at frequent intervals. Influenza virus not only is a human virus
but has a complex relationship that includes replication in several hosts (see
chapter 20). For example, influenza A
virus infects not only humans but also
pigs, birds, horses, mink, and some aquatic
mammals such as seals and whales.
Epidemiologists can classify a given influenza A virus by its antigenic composi-
Influenza virus provides the
classic paradigm of antigenic
shift and drift
tion, usually on the basis of serologic reactions of the two envelope proteins
hemagglutinin (HA or H) and neuraminidase (NA or N). A common
nomenclature simplification is to refer to
combinations of HA and NA as HxNy
(where 1 # x # 14 and 1 # y # 9), e.g.,
H1N1 or H5N2. At least 14 subtypes of
viral hemagglutinin are known in viruses
that infect birds. Three of these subtypes
are present in viruses that can infect humans, and at least two can infect pigs,
horses, and aquatic mammals.
More than nine NA subtypes are
known, and, like HA subtypes, viruses
with these subtypes have a characteristic
host range. Thus, if antisera that recognize
each of these HA and NA subtype proteins
are available, it is possible to trace a virus
population through multiple hosts and its
movement around the world.
1950
1889
H2N2
1900
H3N8
1957
H2N2
1918
H1N1
"Spanish"
?
?
HA
NA
HA
NA
PB2
PB1
PA
HA
NA
NP
M
NS
1968
H3N2
"Asian"
5
8?
H1N1
Postulated evolution of human influenza A viruses from 1889
to 1977. The figure depicts the appearance and transmission of
distinct serotypes of influenza A virus in humans. The bottom
part shows the nature of the avian influenza viruses that reassort
with human viruses. The color of the genome segments represents a particular viral genotype. Segments of the predominant
influenza virus genome and its gene products are indicated in
each human silhouette for each year. The number next to the
arrow indicates how many segments of the viral genome are
known to have been transmitted. The earliest serology data we
have are from 1889 and suggest that H2N2 was the predominant
class in humans. In 1900 the predominant serotype was H3N8.
No data exist for the other influenza virus genes present at these
times, and these segments are not illustrated. Phylogenetic evidence is consistent with the appearance by 1918 of an influenza
virus with eight distinct segments. This virus is thought to be of
avian origin and is characterized by the H1N1 serotype (red). It
was also found in pigs and was carried from North America to Eu-
"Hong Kong"
PB2
PB1
PA
HA
NA
NP
M
NS
3
H2N2
6
PB2
PB1
PA
HA
NA
NP
M
NS
1977
H1N1
"Russian"
PB2
PB1
PA
HA
NA
NP
M
NS
H1N1
H3N2
2
H3N?
rope by U.S. soldiers. It gained notoriety as the cause of the catastrophic Spanish influenza pandemic of 1918. In 1957 the Asian
pandemic was caused by a virus that acquired three genes (PB1,
H2, and N2) from avian viruses infecting wild ducks (yellow) and
retained five other genes from the circulating human strain with
H2N2 (red). As the Asian strains appeared, the H1N1 strains disappeared from the human population. In 1968, the Hong Kong
pandemic virus acquired two new genes (PB1 and H3) from the
wild duck reservoir (blue) and kept six genes that were circulating in human viruses (red and yellow). The pandemic virus had
a characteristic H3N2 serotype. After the appearance of this virus,
the Asian H2N2 strains could no longer be detected in humans.
In 1977 the Russian H1N1 strain that had circulated in humans
in the 1950s reappeared and infected young adults and children.
One theory was that this virus escaped from a laboratory. It has
continued to cocirculate with H3N2 influenza viruses in the
human population. Adapted from R. G. Webster and Y. Kawaoka,
Semin. Virol. 5:103–111, 1994, with permission.
529
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CHAPTER 15
B OX 1 5 . 3
Antigenic shift, not drift, was
the driving force for the five
pandemics of human
influenza during the past
century (1890, 1900, 1918,
1957, and 1968)
Viruses causing these worldwide infections can be typed into HA and NA subtypes (see also Box 15.2). Each pandemic
is characterized by a new combination of
HA and NA:
The
The
The
The
1918
1957
1968
1977
flu was H1N1.
“Asian flu” was H2N2.
“Hong Kong flu” was H1N2.
“swine flu” was H1N1.
These dramatic shifts of H and N
serotypes result from the exchange of
genome segments by mammalian and
avian influenza viruses. In general, influenza A viruses that grow well in birds
are not efficient at infecting humans, and
vice versa. This assertion is currently
under scrutiny, as evidence of direct infection of humans by an avian influenza
virus (H5N1) was documented in 1997.
This H5N1 combination of antigens has
never been observed previously in
human infections despite its occurrence
in virulent avian viruses that have caused
major domestic bird epidemics. Since no
humans have immunity to the H5N1
viruses, the appearance of those viruses in
humans was of major concern. It now appears that these viruses are incapable of
efficient spread in human populations.
Virologists have demonstrated that
certain combinations of H and N are better selected in avian hosts than in humans. An important observation was that
both avian and human viruses replicate
well in certain species such as pigs, no
matter what the H/N composition is. Indeed, the lining of the throats of pigs contains receptors for both human and avian
influenza viruses, providing an environment in which both can flourish. Thus,
the porcine host is a good nonselective
host for mixed infection of avian and
human viruses in which reassortment of
H and N segments can occur, creating
new viruses that can reinfect the human
population.
At first glance, one might think that
this combination of human, bird, and pig
(continued)
Genetic reassortment between avian and human influenza A viruses in swine. Studies
of Italian pigs provide evidence for reassortment between avian and human influenza
viruses. The figure shows how the avian H1N1 viruses in European pigs reassorted with
H3N2 human viruses. The color of the segments of the influenza genome indicates the
origin: blue segments are from avian viruses found in swine in 1979, and red segments
are from human viruses found in swine in 1968. The host of origin of the influenza virus
genes was determined by partial sequencing and phylogenetic analysis. These studies support the hypothesis that pigs can serve as an intermediate host in the emergence
of new pandemic influenza viruses. Adapted from R. G. Webster and Y. Kawaoka, Semin.
Virol. 5:103–111, 1994, with permission.
1979
H1N1
-Avian1985–1989
Reassortants
1968
H3N2
-Human-
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Patterns of Infection: a Delicate Balance
B OX 1 5 . 3
531
(continued)
infections must be extremely rare. However, the dense human populations in
Southeast Asia that come in daily contact
with domesticated pigs, ducks, and domestic fowl remind us that these interactions are likely to be frequent. Indeed,
epidemiologists can show that the 1957
and 1968 pandemic influenza A virus
strains originated in the People’s Republic
of China and that the human H and N
serotypes are circulating in wildfowl populations. In the United States, turkeys as
well as swine may serve as intermediate
hosts for mixing of avian and mammalian
influenza viruses, but direct transfer from
turkeys to humans has not yet been
demonstrated.
Southeast Asia should not be the only
focus of attention, as intense production
of domestic swine and turkeys in Europe
of the MHC-peptide complex to the cell surface. Several
viruses express proteins that block MHC class I function at
various points in the pathway (Fig. 15.7; Table 15.4). The
MHC class I pathway is of obvious importance in immunology, but the existence of many of these MHC-processing or regulatory steps was not known until viral
inhibitors were characterized.
Human cytomegalovirus deserves special mention because it has evolved multiple strategies to prevent MHC
class I molecules from exposing viral antigens on the cell
surface. The human cytomegalovirus US6 protein inhibits
peptide translocation into the endoplasmic reticulum
lumen, although by a different mechanism than the herpes simplex virus ICP47 protein (Fig. 15.7; Table 15.4).
The human cytomegalovirus US3 protein detains MHC
class I proteins in the endoplasmic reticulum by direct interaction, while the US11 and US2 proteins eject MHC
class I molecules from the endoplasmic reticulum into the
cytoplasm, where they are degraded by cellular proteases.
Why human cytomegalovirus encodes so many proteins to
block antigen presentation remains an open question (Box
15.4). One possibility is that multiple gene products act additively or synergistically to effect the block of MHC class I
function. Other ideas are that different proteins are required to block MHC class I function in different cell types
or that the concentrations of viral proteins vary in different cell types.
Human cytomegalovirus and Epstein-Barr virus have
evolved another strategy for avoiding cytotoxic-T-cell destruction. The first indication of this phenomenon was the
observation that Epstein-Barr virus-infected individuals do
not produce cytotoxic T cells capable of recognizing the
viral protein Epstein-Barr virus nuclear antigen 1 (EBNA1). This phosphoprotein is found in the nuclei of latently
infected cells and is the only protein regularly detected in
all malignancies associated with Epstein-Barr virus. T cells
specific for other Epstein-Barr virus proteins are made in
abundance, indicating that Epstein-Barr virus nuclear
and the United States, coupled with the
major migratory paths of wild ducks and
geese, is likely to make these regions centers of interspecies transfer as well.
Ito, T., J. N. S. S. Couceiro, S. Kelm, L. G.
Baum, S. Krauss, M. R. Castrucci, I. Donatelli, H. Kida, J. C. Paulson, R. G. Webster,
and Y. Kawaoka. 1998. Molecular basis for the
generation in pigs of influenza A viruses with
pandemic potential. J. Virol. 72:7367–7373.
antigen 1 must possess some special features. Indeed, this
protein contains a remarkable amino acid sequence consisting of arginine-glycine motifs surrounding an internal
glycine-alanine repeat. The sequence inhibits the host proteasome so that relevant Epstein-Barr virus nuclear antigen 1 peptides are not produced at all. This inhibitory
sequence is nonspecific, for it can be fused to other proteins to inhibit their processing and subsequent presentation of peptide antigens normally produced by them.
Human cytomegalovirus uses a different twist of the
same strategy to block proteasome processing of its major
immediate-early protein. Upon infection, the pp65 kinase,
a viral tegument protein encoded by the UL83 gene, phosphorylates the newly synthesized major immediate-early
protein so that it cannot be processed into antigenic peptides. As a result, peptides of immediate-early protein are
not presented on the surface by MHC class I molecules,
and the infected cell escapes early destruction by cytotoxic
T cells.
Many Other Variables Govern the Result
of Infection
In addition to the parameters described in the preceding
sections, other complex variables can determine the
course and result of infections. Of these, the age and immune status of the host, host population density, host interactions, and environmental conditions are predominant
(see chapter 17 for more details).
Acute Infections
Definition and Requirements
An acute infection is one of the best understood of all viral
infection patterns because it is characteristic of many
viruses that grow well in animals and in cultured cells. By
the term “acute,” we mean rapid production of infectious
virus followed by rapid resolution and clearing of the infection by the host. Acute infections are the typical, ex-
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CHAPTER 15
Cytotoxic T lymphocyte
T-cell receptor
CD8
MCMV gp48
HIV Nef
Infected cell
Lysosome
Viral and cellular proteins
Golgi
MCMV m152
Adenovirus E3 19K
HCMV US3
Proteasome
b2-microglobulin
Peptides
MHC heavy chain
HCMV US6
HSV ICP47
Endoplasmic reticulum
HCMV US11, US2
P
b2-microglobulin
P
Adenovirus E1A
HIV Tat
MHC 1
Nucleus
Figure 15.7 Viral proteins block cell surface antigen presentation by the MHC class I
system. In almost every cell, a fraction of newly synthesized proteins translated in the cytoplasm is targeted to the proteasome, where the molecules are digested into peptide fragments (orange). These peptides are transported to the lumen of the endoplasmic reticulum
(lavender) by the Tap transporters in the endoplasmic reticulum membrane (pink channel). Peptides then bind to a cleft in the newly synthesized MHC class I protein complex
(blue) consisting of an MHC class I heavy chain and a b2-microglobulin chain. The complete peptide-MHC class I complex moves into the Golgi apparatus (light blue) and then
to the cell surface, where it can be recognized by the T-cell receptors on the surfaces of
CD81 T cells that are interacting with the cell. Specific viral gene products block (red bars)
this process at almost every step along the pathway. Green arrows indicate stimulation. In
the nucleus, transcription of MHC class I genes can be blocked by E1A or Tat at the promoter (P), as indicated. HSV, herpes simplex virus; HCMV, human cytomegalovirus;
MCMV, mouse cytomegalovirus; HIV, human immunodeficiency virus.
Degraded by
proteasome
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Patterns of Infection: a Delicate Balance
533
Table 15.4 Viral regulation of MHC class I antigen expressiona
Virus
RNA
Mouse hepatitis virus
Respiratory syncytial virus
HIV-1
HIV-1
DNA
Adenovirus
Epstein-Barr virus
Human cytomegalovirus
Herpes simplex virus
Vaccinia virus
Observed effect or postulated mechanism
Decrease in transcription of specific MHC class I genes
Decrease in MHC class I gene transcription
Tat protein-induced reduction in MHC class I gene transcription
Vpu interferes with an early step in synthesis of MHC class I proteins
E3 19K retains MHC class I in ER; reduced transcription of MHC class I genes induced by E1A proteins
EBNA-4 may block generation of antigenic peptides or their transport from the cytoplasm to the ER;
allele-specific decrease in MHC class I appearance on cell surface
US3 retains MHC class I molecules in the ER; US6 inhibits peptide translocation by Tap (ER luminal
domain); US11 and US2 dislocate MHC class I molecule from the ER lumen to the cytoplasm; UL83
blocks IE-1 peptide presentation
ICP47 binds to Tap transporter and blocks import of peptides into the ER
Lower abundance of MHC class I on cell surface induced by unknown mechanisms
a
Abbreviations: HIV-1, human immunodeficiency virus type 1; ER, endoplasmic reticulum; Tap, transporter of antigenic peptide; EBNA-4, Epstein-Barr virus nuclear antigen 4.
pected course for agents like influenza virus and rhinovirus (Fig. 15.8). These infections are relatively brief,
and in a healthy host, virus particles and virus-infected
cells are completely eliminated (cleared) by the immune
system within days. Nevertheless, an acute infection is an
effective survival strategy for a virus, as some progeny are
invariably available for infection of other hosts before the
infection is resolved (Box 15.5).
Inapparent Acute Infections
It is important to distinguish an inapparent acute infection from an unsuccessful infection. Inapparent infections are successful acute infections that produce no
symptoms or disease. Sufficient virus is produced to maintain the virus population, but the amount is below the
threshold required to produce symptoms in the host. The
usual way an inapparent infection is detected is by a rise
in antiviral antibody concentrations in an otherwise
healthy individual. In a healthy host, most acute infections
B OX 1 5 . 4
Human cytomegalovirus
versus the host MHC class I
system
are inapparent because they are quickly confronted and
cleared by the host immune system. Well-adapted
pathogens often follow this infection pattern, as demonstrated by poliovirus, in which more than 90% of infections are inapparent.
Acute Infections Present Common Public
Health Problems
An acute infection is most frequently associated with serious epidemics of disease affecting millions of individuals
every year (e.g., polio, influenza, and measles). The nature
of an acute infection presents difficult problems for physicians, epidemiologists, drug companies, and public health
officials. The main problem is that by the time people feel
ill or mount a detectable immune response, most acute infections are essentially complete and the virus has spread
to the next host. Such infections can be difficult to diagnose retrospectively or to control in large populations or in
crowded environments (e.g., day care centers, military
Human cytomegalovirus expresses at
least five gene products that act directly to
reduce the function of MHC class I molecules. Surprisingly, both human and
mouse cytomegaloviruses produce a protein that resembles host MHC class I proteins. These proteins form a complex with
b2-microglobulin and contain a peptidebinding groove.
One hypothesis is that these proteins
act as decoys to distract natural killer cells.
When such MHC class I decoy proteins are
displayed on the infected cell’s surface,
natural killer cells may ignore them despite the reduced host MHC class I expression. Evidence supporting and negating
this hypothesis has been published, and it
remains to be seen why these viruses have
their own MHC-like proteins.
An inescapable conclusion is that
MHC protein must present an important
host-virus interface in cytomegalovirus
infections.
Ploegh, H. L. 1998. Viral strategies of immune
evasion. Science 280:248–253.
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CHAPTER 15
Induction of
adaptive
response
Virus
growth
Adaptive
response
Establishment
of infection
Memory
Innate
defenses
Threshold level
of virus required
to activate adaptive
immune response
Duration of infection
Entry of virus
Virus cleared
Figure 15.8 The course of a typical acute infection. Relative virus growth plotted as a
function of time after infection. The concentration of infectious virus increases with time
as indicated by the jagged red line. During the establishment of infection, only the innate
defenses are at work. If the virus reaches a certain threshold level characteristic of the virus
and host (purple), the adaptive responses initiate. After 4 to 5 days, effector cells and molecules of the adaptive response begin to clear the infection by removing virus particles and
infected cells (green). When virus is cleared and infected cells are eliminated, the adaptive
response ceases. Antibodies, residual effector cells, and “memory” cells provide lasting protection should the host be reinfected at a later date. Redrawn from C. A. Janeway, Jr., and
P. Travers, Immunobiology: the Immune System in Health and Disease (Current Biology Ltd. and
Garland Publishing, New York, N.Y., 1996), with permission.
camps, college dormitories, nursing homes, schools, and
offices). Effective antiviral drug therapy requires treatment
early in the infection, often before symptoms are manifested, because by the time the patient feels ill the viral infection has been resolved. Antiviral drugs can be given in
anticipation of an infection, but this strategy demands that
the drugs be safe and free of side effects. Moreover, as we
discuss in chapter 19, our arsenal of antiviral drugs is very
small, and drugs effective for many common acute viral
diseases simply do not exist.
fense against subsequent infections. In immunocompromised individuals, acute infections can be disastrous, primarily because the infection does not remain localized to
the primary site of infection. This property highlights containment of an acute infection as a central role of the immune system in the delicate balance between virus offense
and host defense. As we discuss in chapter 17, the protective immune response that follows acute infection by some
viruses, such as dengue virus, actually makes subsequent
infection by dengue virus of a different serotype much
more severe.
Defense against Acute Infections
The typical immediate host response that limits most acute
infections is the innate response: synthesis of interferons
and lytic attack by natural killer cells. The antiviral defense
strategy of killing a few cells as a “firebreak” is quite common and very effective. In a naive host, the adaptive immune response (antibody and activated cytotoxic T cells)
does not influence virus growth for several days, but it is
essential for final clearance of virus and infected cells from
the infected host, as well as for providing memory for de-
Multiple Acute Infections in a Single Host
An initial acute infection by some viruses may be followed
by a second or third round of infection in the same animal.
In these cases, virus spreads from the primary site of infection to other tissues, where a second acute infection can
occur. We will discuss mechanisms of spread in more detail
in chapter 17. In hematogenous spread, virus particles
enter the bloodstream from the primary site via the lym-
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Patterns of Infection: a Delicate Balance
B OX 1 5 . 5
Uncomplicated acute
infection by influenza virus
An influenza virus infection begins in
the upper respiratory tract by inhalation
of droplets from a sneeze or cough by an
infected individual. Virus replicates in ciliated columnar epithelial cells of the respiratory epithelium, releasing progeny
virus that spreads to nearby cells. Infectious virus can be isolated for 1 to 7 days,
with the peak of released virus occurring
on the fourth or fifth day after infection.
About 48 h after the initial infection,
symptoms appear abruptly such that in-
phatic system or subepithelial blood vessels serving that
site. Viruses can also infect mobile cells that enter the blood
(e.g., lymphocytes) and migrate to new tissues. The first
appearance of virus particles in the blood is called a primary viremia. Once in the circulation, the virus or virusinfected lymphocytes can initiate another acute infection of
internal organs, including the liver, spleen, lungs, or heart.
Progeny virus resulting from this second acute infection
can also enter the bloodstream, producing a secondary
viremia with a potential for even more serious problems
for the host. Secondary viremias are usually characterized
by virus titers in the blood much higher than those of primary viremias. Classic examples of viruses that establish
acute primary and secondary viremias are mousepox,
smallpox, and measles viruses. Virus can also spread from
the primary site of infection to the peripheral and central
nervous systems by infecting neurons in close proximity to
the primary infection (neurological spread). This pattern
of multiple acute infections, viremias, and spread to the
nervous system is characteristic of varicella-zoster virus, an
alphaherpesvirus that causes the familiar childhood disease
chickenpox (Fig. 15.9).
Pathogenic Effects of an Acute Infection
While many symptoms of an acute infection (e.g., fever,
malaise, aches, nausea) are actually due to a vigorous host
immune response, an acute viral infection of internal organs, the gut, the respiratory system, or the nervous system can cause considerable damage because infected cells
are killed by infection and by the immune system. If a sufficient number of cells are infected, severe problems may
result. Symptoms such as diarrhea, poor lung or liver
function, and breakdown of capillary beds or the bloodbrain barrier can arise from direct viral damage to important epithelial and endothelial surfaces of the body. Local
cell damage caused by active replication, by viral proteins,
535
fected individuals can almost pinpoint the
hour that they noticed they had the flu.
Symptoms last for about 3 days and then
begin to abate.
The infection typically resolves within
a week through action of the innate and
acquired immune systems, but it may
take several weeks before the individual
feels completely well because of the
lingering effects of the host defensive
responses.
or by local necrotic reactions also can have serious consequences, including secondary infections by opportunistic
bacteria. As discussed in chapter 17, several viral membrane proteins possess intrinsic activities that may be toxic,
such as the human immunodeficiency virus gp120 envelope protein, which damages neurons. In the case of enteric rotaviruses, a single nonstructural viral protein
(NSP4) alone may be sufficient to cause pathological fluid
loss in gut epithelial cells.
Persistent Infections
Definition and Requirements
Unlike an acute infection, a persistent infection is not
cleared quickly and virus particles or viral products continue to be produced for long periods. Infectious virus may
be produced continuously or intermittently for months or
years (Fig. 15.1). In some instances, viral genomes remain
long after viral proteins can no longer be detected. Distinctions have been made between persistent infections
that are eventually cleared (chronic infections) and
those that last the life of the host (latent infections or slow
infections; see next sections).
Many viruses can establish a persistent infection (Table
15.5). They not only shut down any lethal or cytolytic activities they encode or initiate but also must avoid host antiviral defenses for extended periods. For example, some
arenaviruses, like lymphocytic choriomeningitis virus, are
inherently noncytopathic in their natural hosts and maintain a persistent infection if the host cannot clear the
infected cells. Other viruses, like the herpesvirus EpsteinBarr virus, use alternative transcription and replication
programs to maintain the viral genome in some cell types
with no production of virus particles. In any case, the evasion of host immune defenses is paramount in establishing
a persistent infection.
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Infections of Tissues with Reduced
Immune Surveillance
Infection via conjunctiva
and upper respiratory
tract
Day 0
Replication in primary
lymph nodes
Day 4–6
Primary viremia
Replication in liver,
spleen, and other organs
Secondary viremia
Day 14
Infection of skin and
appearance of rash
Sensory
neurons
Reactivation
Sensory
ganglion
Infection of sensory
ganglia and establishment
of latent infection
Satellite
cells
To central
nervous
system
Figure 15.9 Model of varicella-zoster virus (VZV) infection
and spread. The virus infects the conjunctiva or mucosa of
the upper respiratory tract and then moves to infect regional
lymph nodes. After 4 to 6 days from the initial infection, infected T cells enter the bloodstream, causing a primary
viremia. These infected cells subsequently invade the liver,
spleen, and other organs, causing a second round of infection.
Virus and virus-infected cells are then released into the bloodstream in a secondary viremia, subsequently infecting the
skin. This third round of infection gives rise, after 2 weeks
from the initial infection, to the characteristic vesicular rash of
chicken pox. Following acute replication in the skin, VZV
then infects sensory ganglia of the peripheral nervous system,
where it establishes a new pattern of infection called latency.
The nervous system is not subject to vigorous immune surveillance and is an excellent site for a virus to hide. Later in
life, probably as the specific immune response to VZV wanes,
the latent virus occasionally reactivates and initiates another
acute infectious cycle. This causes the characteristic recurrent
disease called shingles, often accompanied by a painful condition called postherpetic neuralgia.
Cells and organs of the body differ in the degree of their
immune defense. Tissues with surfaces exposed to the environment (e.g., skin, glands, bile ducts, and kidney
tubules) do not employ active immune surveillance, presumably because they are exposed to foreign matter on a
routine basis and possess other mechanisms to contend
with this situation. If a virus can infect such tissue, it may
establish a persistent infection. Other organs, such as the
eye, avoid damaging lymphocyte invasion by expression of
Fas ligand. When invading T cells recognize and bind Fas
ligand on cells of the eye, the T cells die by apoptosis. Certain compartments of the body, including the central nervous system, vitreous humor of the eye, and areas of
lymphoid drainage, are thought to be devoid of effectors of
humoral defense such as the complement system. The eye
and brain may be isolated from routine immune surveillance because they are highly susceptible to damage that
might result from the fluid accumulation, swelling, and
ionic imbalances produced by an inflammatory response.
Moreover, because most neurons do not regenerate, immune defense by cell death is obviously detrimental. Tight
junctions between the epithelial cells that line brain capillaries and ventricles, called the blood-brain barrier, limit
entry of some molecules and cells. Lymphocytes are able
to enter the central nervous system but are not retained
unless they encounter properly presented foreign antigens. However, neurons (as well as muscle and other cells)
do not express MHC proteins readily and in normal circumstances are not “seen” by cytotoxic T cells. As a consequence, the brain is a favored site for the establishment of
a persistent infection by a variety of viruses.
Herpesviruses, papovaviruses, and some complex retroviruses are prime examples of infectious agents that establish persistent infections, in part by infecting tissues with
reduced immune surveillance. By replicating on luminal
surfaces of glands and ducts with poor immune surveillance (kidney, salivary, and mammary glands), human
cytomegalovirus will be shed almost continually in secretions. Possibly the most extreme example of immune
avoidance is represented by papillomaviruses that cause
skin warts. Productive replication of these infectious particles occurs only in the outer, terminally differentiated skin
layer where an immune response is impossible. Dry skin is
continually flaking off, ensuring efficient spread of virus.
One can verify this assertion by running a finger along a
clean surface in the most hygienic hospital and noticing
a white film, which is 70 to 80% keratin from human
skin. Molecular biologists often discover this abundance of
dried skin in the laboratory when examining silver-stained
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Patterns of Infection: a Delicate Balance
537
Table 15.5 Persistent viral infections of humans
Virus
Site of persistence
Rubella virus
Hepatitis C virus
Measles virus
CNSa
Liver
CNS
Human immunodeficiency virus
Human T-cell leukemia virus types 1 and 2
Hepatitis B virus
Hepatitis D virus
Polyomavirus JC
Polyomavirus BK
Papillomavirus
Adenovirus
Varicella-zoster virus
Herpes simplex virus types 1 and 2
Epstein-Barr virus
Human cytomegalovirus
CD41 T cells, macrophages, microglia
T cells
Liver, lymphocytes
Liver
Kidneys, CNS
Kidneys
Skin, epithelial cells
Adenoids, tonsils, lymphocytes
Sensory ganglia
Sensory and autonomic ganglia
B cells, nasopharyngeal epithelia
Kidneys, salivary glands, lymphocytes,
macrophages, stromal cells
Consequence(s)
Progressive rubella panencephalitis
Cirrhosis, hepatocellular carcinoma
Subacute sclerosing panencephalitis, measles
inclusion body encephalitis
AIDS
Leukemia
Cirrhosis, hepatocellular carcinoma
Pathological synergy with hepatitis B virus
Progressive multifocal leukoencephalopathy
Hemorrhagic cystitis
Papillomas, carcinomas
None known
Zoster (shingles), postherpetic neuralgia
Cold sores, genital herpes
Lymphoma, carcinoma
Pneumonia, retinitis
a
CNS, central nervous system.
protein gels; the major band is often contaminating keratin.
Direct Infection of the Immune System Itself
Some viruses infect cells at the very heart of the host antiviral defense system, lymphocytes and macrophages.
Such cells not only play direct roles in the immune defense but also migrate to the extremes of the body, providing easy transport of virus to new areas and hosts.
Infection of such cells is likely to reduce immune function
directly, and as a result virus should be able to persist in
the host. The effectiveness of this strategy is demonstrated
by the surprisingly large number of viruses known to infect lymphocytes and monocytes (Table 15.6).
Human immunodeficiency virus provides a powerful
reminder of how effective infection of the immune system
can be (see also chapter 18). The virus infects not only
CD41 T-helper cells but also monocytes/macrophages that
can transport virus to the brain and other organs. Professional antigen-presenting cells, such as the dendritic cells
in the spleen discussed in chapter 14, are also infected. An
untreated infected individual continues to produce prodigious quantities of virus for years. Disease is characterized
by persistent immune activation. One line of research suggests that very early reactions in virus attachment to susceptible T cells play a major role in this phenomenon.
When the virus attaches to susceptible T-helper cells in tissue culture by binding of envelope gp120 protein to the
Table 15.6 Viruses that infect lymphocytes and monocytes
(1) strand RNA viruses
Poliovirus
Rubella virus
Caliciviruses
(2) strand RNA viruses
Lymphocytic choriomeningitis virus
Measles virus
Mumps virus
Respiratory syncytial virus
Influenza A virus
Vesicular stomatitis virus
Parainfluenza virus
Retroviruses
Murine leukemia virus
Feline leukemia virus
Human T-cell leukemia virus types 1 and 2
Human immunodeficiency virus
Endogenous C-type virus
Single-stranded DNA viruses
Porcine parvovirus
Minute virus of mice
Double-stranded DNA viruses
Hepatitis B virus
Papovaviruses
Group C adenoviruses
Herpes simplex virus
Varicella-zoster virus
Epstein-Barr virus
Human herpesvirus 6
Human herpesvirus 7
Human cytomegalovirus
Leporipoxviruses
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CD4 and Ccr5 chemokine receptors, a signal transduction
cascade is activated. As a result, the infected T cells express
putative chemoattractant molecules that attract more uninfected T cells (new host cells) to the site of infection In
addition, the virus replicates efficiently in these activated
cells. Thus, as a result of engagment of the receptor and
coreceptor, both virus replication and cell-cell spread are
accelerated.
Two Viruses That Cause Persistent Infections
Lymphocytic Choriomeningitis Virus
Lymphocytic choriomeningitis virus, a member of the
family Arenaviridae, was the first virus associated with
aseptic meningitis in humans. Perhaps more importantly,
its study has illuminated fundamental principles of immunology and viral pathogenesis, particularly those that
underlie persistent infection and cytotoxic-T-cell recognition and killing. Lymphocytic choriomeningitis virus is not
cytopathic and also infects both mice and humans. It was
noted early on that the virus spreads from rodents (the
natural host) to humans, in which it can cause severe neurological and developmental damage. Infected rodents
normally produce large quantities of infectious virus,
which is excreted in feces and urine throughout their lives
without any apparent detrimental effect. These mice are
called carriers because of such lifelong production of
virus. The carrier state occurs in part because of two phenomena: the virus is not cytopathic; and, if mice are infected congenitally or immediately after birth, the virus is
not recognized as “foreign” and thus cannot be cleared by
the murine immune system. However, if the virus is injected into the brains of healthy adult mice in the laboratory, the mice die of acute immunopathologic encephalitis.
This disease is similar to that contracted by humans who
develop aseptic meningitis after lymphocytic choriomeningitis virus infection.
From results with the laboratory mouse model, we now
understand that cytotoxic T cells are required both for
clearing lymphocytic choriomeningitis virus and for the
lethal response to intracerebral infections. If adult mice are
depleted of such cells, direct injection of virus into the
brain is no longer fatal. Instead, the mice express infectious virus throughout their lifetimes, precisely as seen in
persistent infections of neonates. When lymphocytic
choriomeningitis virus-responsive, cytotoxic T cells are
added back to neonates with a persistent infection, all
virus is cleared after several weeks. In the case of neonatal
persistent infection, virus is not in the brain, so no immunopathologic encephalitis is promoted by the activated
T cells. How the neonatal infection effectively “silences”
the effector system from clearing virus is currently under
investigation. Recent experiments have implicated an ac-
tive process of clonal deletion of T lymphocytes capable of
recognizing lymphocytic choriomeningitis virus antigens.
Measles Virus
Measles virus provides a provocative example of the
delicate balance that determines the pattern of infection.
Many important questions remain unanswered about how
this human pathogen switches from an acute to a persistent infection and does so despite an active immune
response.
Measles virus, a member of the family Paramyxoviridae,
is a common human pathogen with no known animal
reservoir. The genome organization and replication strategy are similar to those of the rhabdovirus vesicular stomatitis virus. Measles is one of the most contagious human
viruses, with about 40 million infections occurring worldwide each year, resulting in 1 to 2 million deaths. Normally, it causes an acute infection only once in a lifetime
because a single infection routinely protects the individual. Measles virus is a highly adapted human pathogen,
persisting only in populations sufficient to produce a large
number of new hosts (children). Population geneticists
calculate that communities of 200,000 to 500,000 individuals are required in order to maintain measles virus.
The receptor for measles virus is the human complement regulatory cofactor protein CD46. Despite a rather
broad distribution of its receptor, measles virus shows tropism for the respiratory tract, the site of primary infection.
In an acute infection, the disease course runs about 2
weeks—so-called uncomplicated measles (Fig. 15.10). An
acute infection causes cough, fever, and conjunctivitis and,
as already noted, confers lifelong immunity. The familiar
rash is due to a hypersensitivity reaction.
On closer examination, the hallmarks of acute measles
infection are sinister, and as we now appreciate, presage
the subsequent potentially serious problem of persistent
infection. Such hallmarks include the following features:
measles virus kills cells by cell-cell plasma membrane fusion, not by shutting off host macromolecular DNA, RNA,
and protein synthesis; the virus can replicate in a variety
of tissues, including cells of the nervous system and the
immune system; the majority of acutely infected individuals exhibit an abnormal electroencephalograph indicating
subtle neurological damage from uncomplicated measles;
and finally, an initial acute infection results in both virus
dissemination via viremia and general immunosuppression.
Immunosuppression is responsible for many deaths
from secondary infections; it may also be necessary to establish a persistent infection. The immunosuppressive effect is not long-lived, and with proper care the vast
majority of patients recover with no further problems. The
molecular basis for immunosuppression is not yet fully
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Patterns of Infection: a Delicate Balance
A
539
B
Infection
Pleomorphic
particles
100–300 nm
H (hemagglutinin)
Giant cells in infected tissue
F (fusion protein)
Spread
to
Primary viremia
entire
Draining r.e.s.
lymph
nodes
P (phosphoprotein)
Lipid bilayer
Secondary viremia
Virus shedding
Epithelial
necrosis
Spread
to all
body
surface
Polymerase
Antibody
Disease
Koplik spots
RNA = 16 kb
Days after infection
M (matrix protein)
N (nucleocapsid)
0
2
4
6
Incubation
8
10
12
Prodromal
14
16
18
20
Rash
Recovery
Figure 15.10 Infection by measles virus. (A) Diagrammatic representation of the structure of the pleomorphic measles virion. (B) Course of clinical measles infection and events
occurring in the spread of the virus within the body are illustrated. Four clinically defined
temporal stages occur as infection proceeds and are illustrated at the bottom. As virus
spreads by primary and secondary viremia from the lymph node to the entire reticuloendothelial system (r.e.s.) and finally to all body surfaces, characteristic symptoms and clinical findings appear. The timing of typical reactions that correspond to the clinical stages is
shown by the colored arrows. The telltale spots on the inside of the cheek (Koplik’s spots)
and the skin lesions of measles consist of pinhead-sized papules on a reddened, raised area.
They are typical of immunopathology in response to measles virus antigen. Redrawn from
A. J. Zuckerman et al., Principles and Practice of Clinical Virology, 3rd ed. (John Wiley & Sons,
Inc., New York, N.Y., 1994), with permission.
understood, but several mechanisms have been suggested
(see also chapter 17). We know that the virus infects T and
B cells, as well as macrophages, arresting them in the late
G1 phase of the cell cycle. As a result, the infected cells
cannot perform their normal functions. Uninfected lymphocytes can also be suppressed, as demonstrated by
cocultivation with virus-infected cells. Suppression of this
type requires cell-cell contact and expression of both the
viral hemagglutinin (HA) protein and the fusion protein.
When the viral HA binds to CD46 receptor protein, expression of interleukin-12 (Il-12) is suppressed. As a result, the Th1 immune response is not activated efficiently.
On rare occasions, measles virus genomes and antigens
may persist for years in a single individual. The mechanisms responsible are only now being characterized. Paradoxically, these studies indicate that antibodies produced
to check the acute infection play a central role in driving
the virus into persistent infection. While the mechanism
for this unexpected effect in humans is not understood,
some insight has come from studying the effect of antibodies during infection of tissue culture cells. Measles
virus-specific antibodies reduce expression of viral membrane proteins on the cell surface by an unknown mechanism. Because one of these proteins, viral fusion protein,
is responsible for the cell-cell fusion that causes cell death,
exposure to antibodies effectively blocks cell killing and
thereby allows viral persistence.
Measles virus can enter the brain by infecting lymphocytes that traverse the body during the viremia following
primary infection. Such a secondary infection has a number of consequences. One is acute postinfectious encephalitis, which occurs in about 1 in 3,000 infections.
The other is a rare, but delayed and often lethal, brain infection called subacute sclerosing panencephalitis.
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This disease results from a slow infection, a unique variation of a persistent infection (see “Slow Infections” below
and Fig. 15.1). Six to eight years after young adults and
children contract measles, about one in a million develop
subacute sclerosing panencephalitis. It appears that this
disease results when intracellular host proteins in cells of
the central nervous system interfere with acute infection
by inhibiting viral gene expression, especially synthesis of
envelope proteins. Available evidence indicates that components essential for the assembly and budding of mature
infectious virus particles are absent in brains of afflicted
patients. One line of thinking focuses on aberrant expression of the matrix (M) protein as an important participant
in this fatal infection. The matrix proteins of (2) strand
RNA viruses are essential for assembly of particles, and the
M protein is often poorly expressed in persistent paramyxovirus infections. Much remains to be discovered, and important questions remain. Does immunosuppression
during an acute infection facilitate infection in the brain?
Are defects in M protein expression and particle assembly
necessary and sufficient to cause disease, or are they effects of other selection processes in the brain? Do the defects in viral gene expression require long exposure to the
host, or are they caused by host cell proteins that reduce
viral gene expression? Transgenic mice expressing the
human measles virus receptor CD46 are now available and
should allow these important questions to be addressed in
a rigorous and controlled fashion.
made during a latent infection. Viral proteins required for
productive replication may not be produced at all, a pattern exemplified by herpes simplex virus. This virus establishes a latent infection in nondividing neurons, and only
characteristic latency-associated transcript (LAT)
RNAs can be detected in the nuclei. In contrast, several
viral proteins may be required to maintain the latent infection, as is the case for Epstein-Barr virus. This virus establishes latency in B lymphocytes when a specific
transcription program produces at least seven viral proteins needed to replicate and maintain the viral genome as
the latently infected cells divide.
If latency is to have any value as a survival strategy, the
latent virus must have a mechanism for reactivation so
that it can spread to other hosts. Reactivation usually follows trauma, stress, or other insults, conditions that may
mark the host as a poor place to continue the latent infection. In the case of herpes simplex virus, reactivation can
also provide a mechanism for reinfection and establishment of a latent infection in more neurons in the same individual. This is the so-called round-trip strategy: virus
reactivates from neurons and replicates at mucosal surfaces, and the progeny enter more neuronal termini to repopulate ganglia. The latent infection is remarkable for its
simplicity and effectiveness as a survival strategy.
Two Viruses That Produce Latent Infections
Herpes Simplex Virus
Latent Infections
An Extreme Variation of the
Persistent Infection
Latent infections can be characterized by four general
properties: a nonreplicating cell is infected or the viral
genome is replicated in conjunction with host DNA replication so that the cell cycle is not interrupted; immune detection of the cell harboring the latent genome is reduced
or eliminated; expression of productive cycle viral genes is
absent or inefficient; and the viral genome itself persists
intact so that at some later time a productive acute infection can be initiated to ensure spread of its progeny to a
new host (Fig. 15.1). The latent genome can be maintained as a nonreplicating chromosome in a nondividing
neuron (herpes simplex virus), become an autonomous,
self-replicating chromosome in a dividing cell (EpsteinBarr virus), or be integrated into a host chromosome
(adeno-associated virus).
Such “long-term parking” of a viral genome is remarkable for its stability, which requires a delicate balance
among the regulators of viral and cellular gene expression.
Generally, only a restricted set of viral gene products are
Over 40 million people in the United States (about 20%
of the population) harbor latent herpes simplex virus in
their peripheral nervous system and will experience reactivation of their own private virus sometime in their lifetime. Many millions more carry latent herpes simplex
virus in their nervous system but never report reactivated
infections. Herpes simplex virus is a highly efficient
pathogen as demonstrated by its widespread prevalence in
humans, its only known host. No animal reservoirs are
known, although several laboratory animals, including
rats, mice, guinea pigs, and rabbits, can be infected. Such
success results from the high efficiency of both the productive and latent infections in humans. Herpes simplex
virus is unique in establishing latent infections predominately in terminally differentiated, nondividing neurons.
These cells are excluded from some forms of immune surveillance. They neither replicate their DNA nor divide, and
so once established, a viral genome need not replicate to
persist for the life of the neuron. Finally, sensory neurons
are highly connected through synapses and thus serve as
excellent conduits for transport of virus particles to and
from mucosal surfaces and other segments of the nervous
system. The ability of the virus to establish a latent infec-
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Patterns of Infection: a Delicate Balance
tion is an effective survival mechanism, as neither vaccines
nor antivirals can attack the virus in its latent form. Once
infected with these viruses, the host is infected for life—
latency is absolute persistence.
Many details of the molecular aspects of herpes simplex
virus latency await discovery and explanation, but the
general pathway and critical issues are well established.
Virus infects neurons of sensory and autonomic ganglia
following primary rounds of replication in cells of mucosal
or epidermal surfaces (Fig. 15.11). The general outline of
putative regulatory steps necessary for the establishment,
maintenance, and reactivation of a viral infection after
primary infection is shown in Fig. 15.12. A typical primary
infection of a mouse, showing the time course of production of infectious virus and establishment of a latent infection, is seen in Fig. 15.13. One or two weeks after primary
infection of the ganglion, infectious virus can no longer be
isolated—the operational definition of an established latent infection. If the animal survives the primary infection,
establishment of the latent infection is inevitable. The time
frame for this process varies depending on the animal
species, the concentration and genotype of the infecting
virus, and the site of primary infection. It is not clear how
neurons in the ganglia survive the primary infection. We
do not yet know how the viral genes whose products are
necessary to complete the productive infection are turned
off as latent infections are being established. Plausible hypotheses are that terminally differentiated, nondividing
neurons may lack proteins required for viral gene transcription or may contain repressors of transcription not
found in “permissive” cells. Indeed, there is evidence for
both mechanisms. Viral gene products may also serve to
repress viral gene expression. The latent genome persists
as a nonintegrated circular DNA molecule associated with
nucleosomes and is probably tethered to a specific site in
the nucleus (Box 15.6). Some latently infected neurons
express the latency-associated transcripts mentioned
above. They are discussed in detail in chapter 8.
Nonneuronal cells and the immune system play important roles in establishing the pattern of herpes simplex
virus infection. For example, only 10% of the cells in a
typical sensory ganglion are neurons; the remaining 90%
are nonneuronal satellite cells and Schwann cells associated with a fibrocollagenous matrix. These cells are in intimate contact with ganglionic neurons. Some of the
nonneuronal cells are infected during initial invasion of
the ganglion. The peripheral nervous system is accessible
to antibodies, complement, cytokines, and lymphocytes of
the innate and adaptive immune system. In murine models, the immune response to productive infection in the
ganglion actively influences the outcome of primary infection of neurons. For example, cytotoxic T cells that recog-
541
nize viral antigens and passive immunization with antibodies to virus facilitate efficient establishment of latency.
How this is accomplished without death of the infected
neurons remains controversial.
Establishment of a reactivatable latent infection in sensory ganglia that service mucosal surfaces is a particularly
effective means of ensuring virus transmission because
mucosal contact is widespread among affectionate
humans. Virus can be spread both by infection at mucosal
surfaces or by cuts and abrasions in the skin. However, a
person must be actively producing infectious virus to
transfer herpes simplex virus to another person. Local
spread of reactivated virus is rapidly curtailed because the
host is immunized during the primary infection. The ability of herpes simplex virus to spread among mucosal epithelial cells after reactivation in an immune host may be
facilitated by action of the viral protein ICP47, which
blocks MHC class I presentation of viral antigens to the T
cells that initiate the cellular immune response. Such activity may provide the virus with sufficient time for a few
rounds of replication before elimination by activated cytotoxic T lymphocytes.
Some individuals with latent herpes simplex virus experience reactivation every 2 to 3 weeks, while others have only
rare or no episodes of reactivation. The signaling mechanisms that reactivate the latent infection are not well characterized, although sunburn, stress, nerve damage,
depletion of nerve growth factor, steroids, heavy metals,
and trauma (including dental surgery) all promote reactivation. We can imagine that such diverse exogenous signals converge to activate specific cellular proteins needed
for transcription of herpes simplex virus immediate-early
genes and thus activate the productive transcriptional program. Alternatively, reactivation may not require action of
viral transcriptional regulators but may result from activation of cellular analogs of these immediate-early proteins
capable of direct activation of viral early genes (Fig. 15.12).
Reactivation may be an all-or-none process requiring but a
single reaction, such as inactivation of a repressor or production of an activator, to “flip the switch” that triggers the
cascade of gene expression of the lytic pathway. Glucocorticoids are excellent examples of such activators, as they
stimulate transcription rapidly and efficiently while inducing an immunosuppressive response. These properties
explain the observation that clinical administration of glucocorticoids frequently results in reactivation of latent herpesvirus. An interesting hypothesis is that spontaneous
reactivation results from single reactions in individual neurons, each of which leads to a small burst of herpes simplex
virus transcription. When the stimulus is strong enough,
such sporadic transcription ultimately passes a threshold,
resulting in replication and reactivation. This idea is consis-
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Virus
Mucosal surface
Epithelial cells (cell-cell spread)
Sensory innervation
by neurons
Potential spread
to lymph and
circulatory system
Spread to peripheral
nervous system
Sensory neuron cell bodies
Peripheral
axon
Immune
surveillance
Sensory ganglion
of peripheral
nervous system
Central
axon
Satellite cells
To central nervous system
(spinal cord and brain)
Figure 15.11 Herpes simplex virus primary infection of a sensory ganglion. Virus replication occurs
at the site of infection (primary infection), usually mucosal surfaces, and the infection may or may not
be apparent. Host innate defenses normally limit the spread of virus at this stage. Virus may infect local
immune effector cells, including dendritic cells and infiltrating natural killer cells. Virus also spreads locally between epithelial cells and has access to lymph and the circulation as well as to neurons that
service the mucosal surfaces. Virus replication in epithelial cells may not be an absolute requirement
for infection of neurons because replication-defective herpes simplex virus mutants can establish a latent infection in model systems. During the primary infection, virus is taken up at nerve endings of the
local sensory and autonomic nerves innervating the area of infection and is transported to the neuronal cell bodies in the peripheral ganglia by transport systems operating within the neurons. It is likely
that the virus loses its envelope on entry into these neurons and the capsid is transported to the neuronal cell body, where it delivers the viral DNA to the nucleus. In some neurons, virus enters the productive cycle forming many infectious virus particles that then spread to other neurons in synaptic
contact with the infected neuron (transsynaptic spread). Such spread could be reflected in local spread
among cells in the ganglion, more distant spread to other ganglia that are in synaptic contact, or spread
to the central nervous system (CNS; the spinal cord or the brain). Spread to the CNS from the peripheral nervous system is frequently fatal, but fortunately it is a rare consequence of primary herpes simplex virus infection. Primary infection is normally constrained to the ganglion. Viral proteins can also
be detected in the nonneuronal satellite cells of the ganglion during the acute infection. Sensory and
autonomic ganglia are in close contact with the bloodstream and do not have the so-called blood-brain
barrier found in the CNS. The ganglion can become inflamed and be transiently visited by various lymphoid cells that leave the circulatory system in response to the infection. Infection of the ganglion is
usually resolved within 7 to 14 days after primary infection, virus particles are cleared, and a latent infection of some neurons in the ganglion is established (see also Fig. 15.13).
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Patterns of Infection: a Delicate Balance
Viral
activators
543
Primary herpes simplex virus
infection
Cellular activators
Tegument
proteins
Cellular repressors
Immediate-early
gene expression blocked
Immediate-early
genes expressed
?
Early genes expressed
DNA replication
Commitment to productive
growth, inhibition
of apoptosis
Latent infection
established
?
Viral gene
products?
?
Late genes expressed
Particle assembly
DNA packaging
LAT expression
Reactivation
Particle egress
Maintenance
Stimuli
Stress response
Viral and cellular
activator expression
Infectious virus
produced and spread
Latent
infection
Productive
infection
Figure 15.12 General flowchart for establishment, maintenance, and reactivation of a
latent infection by herpes simplex virus. The green box at the top indicates the primary
infection by virus particles at mucosal surfaces. The productive infection is shown by the
pathway on the left, and the latent infection is indicated by the pathway on the right. The
question marks indicate our lack of knowledge concerning expression and function of viral
proteins at the indicated steps. Infectious particles produced by the productive pathway
may infect other cells and enter either the productive or latent pathway as indicated. Reactivation is indicated by the diagonal arrow from the latent infection to the start of the
productive infection. The question marks note the current controversy as to whether reactivation requires “going back to go” (immediate-early gene expression) or expression of
early genes required for in DNA replication. Adapted from M. A. Garcia-Blanco and B. R.
Cullen, Science 254:815–820, 1991, with permission.
tent with the low levels of immediate-early and early transcripts that can be detected in ganglia harboring a latent
viral infection. Currently, we know only that reactivation
of the latent genome requires resumption of transcription
and initiation of viral DNA replication.
Epstein-Barr Virus
Epstein-Barr virus, which infects only humans, is exceptionally efficient at establishing latent infections in B lymphocytes. About 90% of the world’s population carries
Epstein-Barr virus as a latent virus. The virus has two major
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Viral titer (PFU/ganglion)
104
103
102
101
100
0
2
4
6
Time (days)
8
10
12
Figure 15.13 Replication of infectious herpes simplex virus
type 1 in mouse trigeminal ganglia during acute infection.
Mice were anesthesized and infected by a standard strain of
herpes simplex virus by dropping approximately 105 plaqueforming units of virus onto the cornea of one eye that had
been lightly scratched with a sterile needle. After a few minutes, the liquid was blotted and the animal was allowed to recover. At selected time points, animals were euthanized and
the trigeminal ganglia were dissected quickly and frozen.
Each point on the graph (red line) represents the geometric
mean titer in plaque-forming units from eight individual ganglia from two different experiments tested at the indicated
time after infection. Uninfected animal controls are indicated
by the blue line.
B OX 1 5 . 6
Neurons harboring latent
herpes simplex virus often
contain hundreds of viral
genomes
target tissues in vivo, B lymphocytes and epithelia. People
carrying latent virus in their B cells not only maintain cytotoxic T cells directed against proteins made by the virus
during the latent infection but also shed small quantities of
infectious virus. How latency is maintained in the face of an
active immune response is an important question.
The primary site of Epstein-Barr virus infection is the
oropharyngeal cavity. The virus probably replicates in differentiating epithelial cells. Children and teenagers are
commonly afflicted, usually following close oral contact
(hence the name “kissing disease”). This acute infection
requires expression of most of the genes contained in the
viral genome. Spread of virus to B cells in an individual
with a normal immune system can induce substantial immune and cytokine responses, resulting in a disease called
infectious mononucleosis. Epstein-Barr virus establishes a latent infection in B cells by an active process that
requires the expression of a specific subset of viral genes.
An important property of one class of virus-infected B cells
is their ability to proliferate indefinitely (i.e., they are “immortalized”). The latent virus genome is maintained as a
circular episome that replicates via a program distinct from
the one used during productive replication, which was
discussed in chapter 9. B cells latently infected by EpsteinBarr virus contain a set of nuclear proteins, termed Epstein-Barr virus nuclear antigens, as well as two
membrane proteins that are important in altering the
properties of the host cells (Table 15.7).
There are at least two distinct phenotypes of viral latency distinguished by the viral gene products made in an
infected B cell. First, a B-cell growth-promoting program is
The number of neurons in a ganglion
that will ultimately harbor latent genomes
depends on the host, the virus, the concentration of infecting virus, and the conditions at the time of infection. It is
possible to infect as few as 1% to as many
as 50% of the neurons in a ganglion. In
the mouse trigeminal ganglion (about
20,000 neurons per ganglion), this means
that less than 200 or more than 10,000
neurons may carry latent genomes. In
controlled experiments in mice, the number of latently infected neurons increases
as the titer of infecting virus increases.
Interestingly, many infected neurons
contain multiple copies of the latent viral
genome varying from fewer than 10 to
more than 1,000; a small number have
more than 10,000 copies. This variation
in copy number has been enigmatic. Does
it reflect multiple infections of a single
neuron or is it the result of replication in
a stimulated permissive neuron after infection by one particle? If it is the latter,
how does the neuron recover from what
should be an irreversible commitment to
the productive cycle?
Recent experiments indicate that
viruses that cannot replicate or whose
replication is blocked by antiviral drugs
exhibit a significant reduction in the
number of latently infected neurons with
multiple genomes. Thus, it is likely that a
single neuron can be infected by multiple
viruses, each of which participates in the
latent infection.
Sawtell, N. M. 1997. Comprehensive quantification of herpes simplex virus latency at the single-cell level. J. Virol. 71:5423–5431.
8938_CH15_518-551.qxd 11/4/99 2:42 PM Page 545
Patterns of Infection: a Delicate Balance
active during acute infection, and these infected B cells express all the known latency-associated genes (sometimes
called stage 3 latency) (Table 15.7). The multiple viral proteins produced are required to establish the latent infection and to avoid elimination of infected cells by the
immune system. A second phenotype is found in which
only EBNA-1 protein is expressed (sometimes called stage
1 latency). Cells of this phenotype are found in Burkitt’s
lymphoma but have been difficult to detect in virus-infected individuals. Recent evidence indicates that major
sites of viral persistence in the peripheral blood are resting
B cells. Such nonreplicating cells express an mRNA that
specifies latent membrane protein 2A but not Epstein-Barr
virus nuclear antigen 1. These cells do not express the B7
coactivator receptor on their surfaces (see chapter 14) and
thus are not killed by cytotoxic T cells.
A remarkable feature of Epstein-Barr virus persistence
is the equilibrium established between active immune
elimination of infected cells and viral persistence. Although normal humans infected with virus maintain cytotoxic T cells directed against many of the viral proteins
synthesized by latently infected B cells, these cells are not
eliminated. This is because some proteins, like latent
membrane protein 1, inhibit apoptosis or immune recognition of the latently infected cells. Moreover, peptides of
Epstein-Barr virus nuclear antigen 1 are not presented to
T cells as discussed above. When the equilibrium between
latently infected B-cell proliferation and the immune response is altered (e.g., after immunosuppression of Epstein-Barr virus-positive patients following bone marrow
transplantation), the virus-immortalized B cells can form
lymphomas (see chapter 16).
545
The signals that reactivate latent Epstein-Barr virus infection in vivo are not well understood, but considerable
information has been obtained by studies of activation of
latent B-cell infections in cultured cells. In these circumstances, activation can be achieved by stimulating certain
signal transduction cascades or by providing an essential
virus transcriptional activator, Zta (Z or zebra protein [see
below]). These studies are all consistent with the hypothesis that the latent state is not maintained by a repressor of
productive infection.
Many signal transduction pathways efficiently reactivate Epstein-Barr virus from the latent state. They can be
activated by diverse interactions, including clustering of
the B-cell antigen receptor CD21 induced by antiimmunoglobulin antibodies (activation of tyrosine kinases), binding of phorbol esters (stimulation of protein
kinase C), and introduction of calcium ionophores. Therefore, it is surprising that latent virus infection of B cells is
so stable in vivo. We now know that virus-encoded latent
membrane protein 2A makes an important contribution to
maintaining the latent infection. This protein inhibits tyrosine kinase signal transduction pathways. It is the first
example of a viral protein that blocks reactivation of a
virus. As virus reactivates efficiently in vivo, a second signal transduction pathway that bypasses the latent membrane protein 2A block must exist, but thus far such a
pathway has not been found.
B cells harboring latent Epstein-Barr virus genomes can
also be reactivated by superinfection with a virus that expresses a virus immediate-early gene product called Zta.
While the role of viral superinfection in virus reactivation
in vivo is not well understood, this phenomenon has pro-
Table 15.7 Epstein-Barr virus proteins required for establishment and maintenance of latent infection
Epstein-Barr
virus proteina
EBNA-1
EBNA-2
EBNA-LP
EBNA-3A and EBNA-3C
LMP-1
LMP-2
a
Function(s)
Maintains replication of the latent Epstein-Barr virus genome during the S phase of the cell cycle. It is a sequencespecific DNA-binding protein and binds to a unique origin of replication called oriP that is distinct from the origin used in the productive replication cycle.
A transcription factor that coordinates Epstein-Barr virus and cell gene expression in the latent infection by
activating the promoters for the LMP-1 gene and cellular genes like CD23 (low-affinity immunoglobulin E Fc
receptor), CD21 (the Epstein-Barr virus receptor, CD23 ligand, and receptor for complement protein C3d)
Required for cyclin D2 induction in primary B cells in cooperation with EBNA-2
Play important roles early in establishment of the latent infection
An integral membrane protein required to protect the latently infected B cell from the immune response. LMP-1
stimulates the expression of several surface adhesion molecules in B cells, a calcium-dependent protein kinase,
and the apoptosis inhibitor Bcl-2.
An integral membrane protein required to block activation of the src family signal transduction cascade; an
inhibitor of reactivation from latency. Two spliced forms exist: LMP-2A and LMP-2B. LMP-2B lacks a receptor
binding domain and may act to modulate LMP-2A.
Abbreviations: EBNA, Epstein-Barr virus nuclear antigen; LMP, latent membrane protein.
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CHAPTER 15
vided significant insight into the action of Zta. The pattern
of gene expression after exposure to this protein is remarkably similar to that seen following reactivation by
phorbol esters. Indeed, as described in chapter 8, Zta functions in part by increasing transcription of virus lytic
genes. This protein also represses the latency-associated
promoters and is responsible for recognition of the virus
lytic origin of replication.
In contrast to the nonpathogenic latent state of herpes
simplex virus, the latent state of Epstein-Barr virus is implicated in several important diseases, including infectious
mononucleosis and at least two kinds of tumor, Burkitt’s
lymphoma and nasopharyngeal carcinoma.
Slow Infections
Sigurdsson’s Legacy: Icelandic Sheep and
Fatal Degenerative Diseases
Many fatal brain diseases characterized by ataxia or dementia stem from another extreme variation of persistent
infection, a pattern called slow infection (Fig. 15.1). It may
be years from the time of initial contact of the infectious
agent with the host until the appearance of recognizable
symptoms. Elucidating the molecular mechanisms responsible for an infectious disease process of such long duration
is a formidable challenge. Experimental analysis of these
unusual diseases began in the 1930s when a flock of
Karakul sheep was imported from Germany to Iceland,
where they infected the native sheep, causing a disease
called maedi/visna. Thanks to the many years of careful
work by Bjorn Sigurdsson and colleagues, we now know
that the maedi/visna syndrome is caused by a lentivirus
quite similar to human immunodeficiency virus. The striking feature that Sigurdsson discovered is the slow progression to disease after primary infection—often more than
10 years. He developed a framework of experimentation
for studying the slow, relentless, usually progressive and
fatal brain infections, including those now proposed to be
caused by prions.
B OX 1 5 . 7
Transmissible Spongiform
Encephalopathies (TSEs)
Slow Viruses and “Unconventional Agents”
Many slow infections are caused by well-known viruses,
including lentiviruses, flaviviruses, rubiviruses, rhabdoviruses, and paramyxoviruses. Slow viral diseases include subacute sclerosing panencephalitis caused by
measles virus (see “Measles Virus” above), tick-borne encephalitis caused by Russian spring-summer encephalitis
virus, progressive rubella panencephalitis, human T-cell
lymphotropic virus type 1-mediated tropical spastic paraparesis, and dementia produced after human immunodeficiency virus infection. While the causative viruses have
been identified, we have much to learn about the molecular mechanisms that promote the slow but relentless progression to disease.
The identity of infectious agents mediating one group of
slow diseases, called transmissible spongiform encephalopathies (TSEs), remains controversial. These
diseases are fatal neurodegenerative disorders afflicting
humans and other mammals (Box 15.7). Some of the controversy stems from the varied interpretation of experiments concerning the physical nature of the infectious
agent. Some investigators contend that TSEs are caused by
viruses or viruslike particles, whereas others propose that
they are caused by infectious proteins called prions.
Human TSE
Initially, the human TSEs were not thought to be transmissible, but studies of the human disease called kuru, a
fatal encephalopathy found in the Fore people of New
Guinea, by Carleton Gajdusek and colleagues proved otherwise. Kuru spread among women and children by ritual
cannibalism of the brains of deceased relatives. When cannibalism stopped, so did kuru. Insightful comparison of the
pathology of scrapie-infected (see below) and kuru-infected brains by William Hadlow led to experiments
demonstrating the transmission of kuru disease from humans to chimpanzees and other primates. Thus, since
1957, the TSE diseases of animals and humans have been
TSE diseases of animals
Scrapie in sheep and goats
Transmissible mink encephalopathy
(TME)
Chronic wasting disease (CWD) (deer,
elk)
Bovine spongiform encephalopathy
(BSE) (“mad cow disease”)
Feline spongiform encephalopathy
(FSE) (domestic and great cats)
Exotic ungulate encephalopathy
(EUE) (nyala and greater kudu)
TSE diseases of humans
Kuru
Creutzfeldt-Jakob disease (CJD)
Fatal familial insomnia (FFI)
Gerstmann-Sträussler syndrome
(GSS)
Prusiner, S. B. (ed.). 1996. Prion diseases. Semin.
Virol. 7:157–223.
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Patterns of Infection: a Delicate Balance
considered to have common features. In humans, TSE diseases fall into three classes, infectious, familial, and sporadic, distinguished by how the disease is initially acquired
(Box 15.8).
Hallmarks of TSE Pathogenesis
For most TSEs, the presence of an infectious agent can
be detected definitively only by injection of organ homogenates into susceptible recipient species. Clinical signs
of infection commonly include cerebellar ataxia (defective
motion or gait) and dementia, with death occurring after
months or years. The infectious agent first accumulates in
the lymphoreticular and secretory organs and then spreads
to the nervous system. In model systems, spread of the
disease from site of inoculation to other organs and the
brain appears to require B cells. The disease agent then appears to invade the peripheral nervous system and spreads
from there to the spinal cord and brain. Once in the central nervous system, the characteristic pathology includes
severe astrocytosis, vacuolization (hence the term “spongiform”), and loss of neurons. Occasionally, dense fibrils or
aggregates (sometimes called plaques) can be detected in
brain tissue at autopsy. There is little indication of inflammatory, antibody, or cellular immune responses. The time
course, degree, and site of cytopathology within the central nervous system are dependent on the particular TSE
agent and the genetic makeup of the host.
Identification of the Scrapie Agent
One of the best studied TSE diseases is scrapie, socalled because infected sheep tend to scrape their bodies
on fences so much that they rub themselves raw. A second
characteristic symptom, tremors caused by skin rubbing
over the flanks, also led to the French name for the dis-
B OX 1 5 . 8
Characteristics of the TSEs
547
ease, tremblant du mouton. Soon after, motor disturbances
appear as a wavering gait, staring eyes, and paralysis of the
hindquarters. There is no fever, but infected sheep lose
weight and die, usually within 4 to 6 weeks of the first appearance of symptoms. Scrapie has been recognized as a
disease of European sheep for more than 200 years. It is
endemic in some countries, like Great Britain, where it affects 0.5 to 1% of the sheep population per year.
Sheep farmers discovered that animals from diseased
herds could pass the affliction to a scrapie-free herd, suggesting that an infectious agent was involved. In 1936, infectivity from extracts of scrapie-affected sheep brains was
shown to pass through filters with pores small enough to
retain all but viruses. In the 1970s, ultracentrifugation
studies indicated that the agent was heterodisperse, and it
could not be banded in density gradients. Even to this day,
purification of the infectious agent to homogeneity has not
been achieved.
Biological Assay of the Scrapie Agent
The only assay for biological activity relies on animal infection. The endpoint assay for infectivity (the highest dilution capable of infecting 50% of the animals, also called
the 50% infective dose [ID50]) is notoriously difficult, requiring 2 to 8 months under the best of conditions (i.e.,
with laboratory-adapted agents) or years (i.e., assaying
primary isolates). Nevertheless, endpoint dilution is a
highly sensitive assay. However, accuracy is a problem,
and typical errors range from 5- to 10-fold. Thus, it is impossible to distinguish 10 and 100% infectivity with confidence. In addition, as deduced from sedimentation
analyses, the infectious agent forms variable aggregates
that confound accurate measurement of infectivity.
An infectious TSE is exemplified by
kuru and iatrogenic spread of disease to
healthy individuals by transplantation of
infected corneas, the use of purified hormones, or transfusion with blood from
patients with Creutzfeldt-Jakob disease
(CJD). Recently, the epidemic spread of
bovine spongiform encephalopathy (BSE,
or “mad cow disease”) among British cattle may have resulted from the practice of
feeding processed animal by-products to
cattle as a protein supplement. There is
continued concern that consumption of
BSE-infected beef will transmit bovine
TSE to humans.
Sporadic CJD is a disease affecting 1
million to 2 million people worldwide,
usually late in life (from age 50 to 70). As
the name indicates, the disease appears
with no warning or epidemiological indications. Kuru may have originally been
established in the small population of
Fore people in New Guinea by eating the
brain of an individual with sporadic CJD.
Familial TSE is associated with an
autosomal dominant mutation in the PrP
gene (see below). Familial CJD, for example, in contrast to sporadic CJD, is an inherited disease.
The important point is that diseases of
all three classes usually can be transmitted experimentally or naturally to primates by inoculation or ingestion of
infected tissue.
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CHAPTER 15
Physical Nature of the Scrapie Agent
A major point of contention is the physical nature of
the infectious agent. Studies as early as 1966 showed that
scrapie infectivity was considerably more resistant than
most viruses to ultraviolet (UV) and ionizing radiation. For
example, the scrapie agent is 200-fold more resistant to UV
irradiation than polyomavirus and 40-fold more resistant
than a mouse retrovirus. Other TSE agents exhibit similar
UV resistance. The scrapie agent is also more resistant to
chemicals, such as 3.7% formaldehyde, and autoclaving
routinely used to inactivate viruses. It is possible to reduce
infectivity by 90 to 95% after several hours of such treatments, but some residual infectivity remains. The inability
to inactivate all infectivity has been used to infer novel
properties for the agent. However, at this level of analysis,
the resistant residual fraction provides no information
about the physical properties of the majority fraction that
was sensitive. On the basis of relative resistance to UV irradiation, some investigators have argued that TSE agents
are viruses well shielded from irradiation or especially efficient in nucleic acid repair. Others have claimed that TSE
agents have little or no nucleic acid at all. Suffice it to say
that TSE agents are not typical infectious agents.
Prions and the PrP Gene
The unconventional physical attributes and slow infection pattern of TSE agents have prompted many to argue
that these agents are not viruses at all. For example, in
1967 the mathematician J. S. Griffith made three suggestions as to how scrapie may be mediated by a host protein,
not by a nucleic acid-carrying virus. His thoughts were the
first of the “protein-only” hypotheses to explain TSE.
In 1981 an important experimental observation was
made by P. Mertz et al., who described scrapie-associated
fibrils in infected brains. This work, and previous studies
indicating that the scrapie agent could be concentrated by
centrifugation, led to the development by Stanley B.
Prusiner and colleagues of an improved bioassay, as well as
a fractionation procedure that allowed the isolation of an
unusual protein from scrapie-infected tissue. This protein
is insoluble and relatively resistant to proteases. Sequence
analysis led to the cloning of a gene called PrP, which is
highly conserved in the genomes of many animals including humans. The PrP gene is now known to be essential
for the pathogenesis of common TSEs.
The PrP gene encodes a 35-kDa membrane-associated
glycoprotein expressed widely in the brain. The protein
can adopt several topological forms in the endoplasmic
reticulum. It can be anchored in the membrane with a
type I or a type II orientation via a transmembrane domain, it can be anchored to the membrane via a phosphatidylinositol linkage, or it can be secreted into the
lumen. The three-dimensional structure of a portion of the
mouse PrP protein has been determined; the amino-terminal half of the protein forms a random coil, while the remaining protein consists of three a-helices and a short
b-pleated sheet. Current evidence indicates that at least
one form of PrP protein binds copper and is sequestered in
caveolae, unique detergent-resistant membrane vesicles
enriched in cholesterol. Despite such basic information,
the function of PrP protein and the role of the various
topological forms remain unknown. Mice lacking the PrP
gene develop normally and have few obvious defects that
can be directly attributed to lack of PrP. At least 18 specific
mutations in the human PrP gene are associated with familial TSE diseases. Furthermore, specific PrP mutations
appear be associated with susceptibility to different strains
of TSE (see below).
Prusiner named the scrapie infectious agent a prion
(an anagram of “proin,” from “proteinaceous infectious
particle”) and proposed that an altered form of the PrP
protein caused the fatal encephalopathy characteristic of
scrapie disease. Occasionally, the term “prion” is used by
some investigators as a synonym for the infectious agent
and for PrP protein. Adriano Aguzzi and Charles Weissman provide a useful definition: a prion is the agent of TSE
with unconventional properties. The term does not have
structural implications other than that a protein is an essential component.
Prusiner’s protein-only hypothesis holds that the essential pathogenic component is an altered conformation of
the host-encoded PrP protein, called PrPsc (“PrP-scrapie”;
also called PrPres for “protease-resistant form”). Furthermore, in the simplest case, PrPsc is proposed to have the
property of converting normal PrP protein into more
copies of pathogenic PrPsc. An important finding in this
regard is that mice lacking both copies of their PrP gene are
resistant to infection. In recognition of his work on this
problem, Prusiner was awarded the Nobel Prize in physiology or medicine in 1997.
Strains of Scrapie Prion
As a result of many serial infections with infected sheep
brain homogenates of different strains of mice and hamsters, investigators have derived distinct strains of scrapie
prion distinguished by length of incubation time before the
appearance of symptoms, brain pathologies, relative abundance of various glycoforms of PrP protein, and electrophoretic profiles of protease-resistant PrPsc. Some
strains also have a different host range. For example,
mouse-adapted scrapie prions cannot propagate in hamsters, but hamster-adapted scrapie prions can propagate in
mice. Sue Priola and Bruce Chesebro discovered that a single amino acid substitution in the hamster protein enables
it to be efficiently converted by mouse PrPsc into hamster
PrPsc. Thus, the barrier to interspecies transmission is in
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Patterns of Infection: a Delicate Balance
the sequence of the PrP protein. Bovine spongiform encephalopathy prions have an unusually broad host range,
infecting a number of meat-eating animals, including domestic and wild cats, and humans. A striking finding is that
different scrapie strains can be propagated in the same inbred line of mice yet maintain their original phenotypes.
Stable inheritance suggests to some investigators that a nucleic acid must be an essential component and has been
used as support for the existence of a virus in TSE disease.
The protein-only hypothesis explains the existence of
strain variation by postulating that each strain represents a
unique conformation of PrPsc. Each of these distinctive
pathogenic conformations is then postulated to convert the
normal PrP protein into a conformational image of itself.
TSE Research Directions
This is an exciting time in TSE research, as many groups
are formulating testable predictions of hypotheses that
TSEs are caused by viruses or by infectious proteins. The
relationship of the protease-resistant form of PrP and PrPenriched fibrils to the disease process remains to be discovered. Several neurodegenerative diseases, including
Alzheimer’s, Parkinson’s, and Huntington’s diseases, are
associated with deposits of insoluble aggregates of cellular
proteins in the brain. These proteins, like PrP, have poorly
structured native conformations which can be destabilized
further by genetic mutations to adopt b-sheet structures.
Perhaps aberrant protein folding is a common feature of
these diseases. Even so, we have little understanding of
how altered or aggregated PrP participates in the fatal
pathogenesis of TSE. Similarly, we do not understand the
molecular nature of the species barrier that exists for
transmission of some prions. We do not yet have a firm
understanding of the role of the immune system in TSE
disease. For example, normal mice do not develop an immune response against the normal or pathological form of
PrP. However, mice lacking the PrP gene develop an immune response to PrP. In fact, most good monoclonal antibodies against PrP are made in mice lacking the PrP gene.
The normal tolerance to PrP may be important for the persistence of PrPsc and its pathological effects.
While most investigators find the protein-only hypothesis compelling, a few maintain that the PrP protein is not
the only participant in the TSE story and that an unrecognized virus is responsible for infection. In their view, the
PrP protein would be a cofactor, a receptor, or a protein
that determines susceptibility to infection. The experiment
that would certainly resolve the basic controversy would
be to produce the putative pathogenic conformation of PrP
in vitro and demonstrate that it causes a specific TSE.
The existence of TSE as a human genetic disease and
the presence of prions in the human and animal food
supply are causes for concern, for we have little under-
549
standing of either the molecular nature of the infectious
agent or the processes of pathogenesis. At a minimum, effort must be focused on basic research toward protection of
the food supply and finding targets for therapeutic intervention.
Other Patterns of Viral Infections
Abortive Infections
An abortive infection is one in which virus infects a susceptible cell or host but does not complete replication,
usually because an essential viral or cellular gene is not expressed. When the host cell is defective for a necessary
viral cofactor, it is said to be a nonpermissive cell, as opposed to a resistant cell, which lacks specific viral receptors
on the cell surface.
Clearly, an abortive infection is nonproductive with respect to making more infectious virus. Even so, it is not
necessarily benign for the infected host. Viral interactions
at the cell surface and subsequent uncoating can initiate
membrane damage, disrupt endosomes, or activate signaling pathways that cause apoptosis and interferon production. For some viruses, an abortive infection may proceed
far enough for viral early proteins to be made so that the
infected cell is recognized by cytotoxic T cells. Such an infection would then induce an interferon, as well as an inflammatory response which may be damaging to the host
if sufficient cells are so infected. Recently, it was discovered that the human immunodeficiency virus structural
protein Vpr can damage cells when it is associated with
virions containing noninfectious genomes. Vpr is required
for the infection of nondividing cells such as macrophages,
and it can induce cell cycle arrest in the G2 phase. When
these noninfectious viruses bind to T cells, the cells arrest
in the G2 phase. It has been suggested that most of the particles in an infected individual are noninfectious, but because of the presence of virion Vpr, these particles
participate in immune suppression due to loss of T-cell
function.
With the advent of modern viral genetics, virologists
can construct defective viruses which, in the absence of a
complementing gene product, initiate an abortive infection. One popular idea is to use such “synthetic” defective
viruses as vectors to deliver genes for gene therapy or vaccine production. To be effective, cytopathic genes of a
prospective viral vector must be eliminated. Many of these
nonlethal viral vectors are missing essential genes and are
designed to express only the therapeutic cloned gene after
infection. Care must be taken to ensure that the infection
is truly noncytotoxic. Cytotoxicity and inflammatory host
responses are of particular concern if the therapeutic gene
is to be delivered to a substantial number of cells, a process
that requires administration of many virus particles.
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Transforming Infections
A transforming infection is a special class of persistent infection. A cell infected by certain DNA viruses or retroviruses may exhibit altered growth properties and begin to
proliferate faster than uninfected cells. Often this change is
accompanied by integration of viral genetic information.
Virus particles may no longer be produced, but some or all
of their genetic material generally persists. We characterize this pattern of persistent infection as transforming
because of the change in cell behavior. It is also considered
oncogenic because transformed cells can cause cancer in
animals. This important infection pattern will be discussed
in detail in chapter 16.
Perspectives
The patterns of infection confront us with several questions that challenge our basic understanding of virology. A
particular infection pattern can be a defining characteristic
of a virus family, yet why this should be is not always obvious. Why has one particular pattern been selected over
another? As we discuss in chapter 20, virus evolution occurs when new virus populations emerge from selection
pressures. What are the selective advantages or disadvantages of a given infection pattern? Is an acute infection any
better as a survival strategy than a persistent infection?
How do we rationalize the characteristic pattern of infection in terms of pathogenesis (the spectrum of events
leading to disease)?
One hypothesis is that successful viruses establish an infection pattern that results in benign symbiosis with
their hosts. In this case, a successful virus neither helps nor
harms the host. Lewis Thomas sagely wrote that pathogenesis is an aberration of symbiosis, an overstepping of
boundaries. Another hypothesis is that a successful virus
need not have a static or benign relationship with its host.
Rather, virus and host populations are in constant flux,
and the successful relationship is better described as an approach to equilibrium (benign symbiosis may never be attained). In this case, viruses engage in a contentious
relationship with the host population, meeting defensive
measures with countermeasures. The relationship may
harm individuals in the host population in the short run to
achieve long-term survival of the virus population. In this
model, pathogenesis may be a necessary survival feature
of the virus and would be selected during evolution of the
relationship. We will continue the discussion of viral
pathogenesis in more detail in chapter 17.
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